Method of producing a temporally spaced subtracted (TSS) cDNA library and uses thereof

ABSTRACT

This invention provides a method for producing a temporally spaced subtracted cDNA library comprising: a) isolating temporally spaced RNAs from cells; b) generating cDNA inserts from the RNAs isolated from step (a); c) producing a temporally spaced cDNA library having clones containing the cDNA inserts generated from step (b); d) producing double stranded cDNA inserts from the temporally spaced cDNA library; e) denaturing the double stranded cDNA inserts; f) contacting the denatured double stranded cDNA inserts produced in step (e) with single-stranded DNAs from another cDNA library under conditions permitting hybridization of the single-stranded DNAs and the double-stranded cDNA inserts; g) separating the hybridized cDNA inserts from the unhybridized inserts; h) generating a cDNA library of the unhybridized inserts, thereby generating a temporally spaced subtracted cDNA library.

[0001] This application is a continuation-in-part of U.S. Ser. No.08/774,465 filed Dec. 30, 1996, the contents of which are herebyincorporated by reference into the present application.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] The invention disclosed herein was made with Government supportunder National Cancer Institute Grant No. CA35675. Accordingly, the U.S.Government has certain rights in this invention.

[0003] Throughout this application, various references are referred towithin parentheses. Disclosures of these publications in theirentireties are hereby incorporated by reference into this application tomore fully describe the state of the art to which this inventionpertains. Full bibliographic citation for these references may be foundat the end of this application, preceding the claims.

BACKGROUND OF THE INVENTION

[0004] Terminal differentiation in human melanoma cells correlates withtemporal changes in the expression of specific target genes. To definethose genes that may be critical for this process a subtractionhybridization approach was used. cDNA libraries were constructed fromactively proliferating H0-1 human melanoma cells (driver cDNA library)and cultures treated for various time periods with the combination ofrecombinant human fibroblast interferon (IFN-β) and mezerein (MEZ)(temporally spaced tester cDNA library) that induces terminaldifferentiation (Jiang and Fisher, 1993). From these two cDNA libraries,an H0-1 IFN-β+MEZ temporally spaced subtracted (TSS) cDNA library wasconstructed. Random screening of this TSS cDNA library identifies cDNAsthat display differential expression as a function of induction ofgrowth arrest and terminal differentiation, called melanomadifferentiation associated (mda) genes. In the present study theproperties of the novel mda-9 gene were analyzed. This cDNA encodes aunique protein of 298 amino acids with a predicted size of 32 to ˜34kDa. Southern blotting analysis indicates that mda-9 is an evolutionaryconserved gene. Tissue distribution analysis documents comparableexpression in fifty human tissues, with slightly elevated expression inbrain (putamen) and spleen (adult and fetal). Treatment of H0-1 humanmelanoma cells with IFN-β+MEZ results in a biphasic induction of mda-9with maximum expression 8 and 12 h post-treatment and reduced expressionat 24 h. In terminally differentiated and irreversibly growth arrestedhuman melanoma cells, the level of mda-9 mRNA is reduced. Thesuppression in mda-9 expression is not simply a function of growthinhibition, since treatment of H0-1 cells with interferons, includingIFN-β, leukocyte interferon (IFN-α) or immune interferon (IFN-γ),elevates mda-9 expression even though they suppress growth. Thesestudies demonstrate that subtraction hybridization using temporallyspaced RNA samples, resulting in a TSS cDNA library, can identify genes,such as mda-9, that are down-regulated during terminal celldifferentiation in human melanoma cells. Further studies are necessaryto define the precise role of mda-9 in the process of terminaldifferentiation.

[0005] Cancer is a progressive disease characterized by both qualitativeand quantitative changes in the phenotypes of evolving tumor cells(1-5). Although cancer can develop as a consequence of single ormultiple genetic alterations, a common theme in carcinogenesis involvesabnormal programs of differentiation (6-10). Attempts to exploit thisdefective differentiation process in cancer cells has led to thedevelopment of a therapeutic approach called ‘differentiation therapy’(6-11). This strategy is based on the use of single or multiple agentsthat induce cancer cells to become more differentiated with aconcomitant reduction or loss of growth potential (6-12). In order toutilize differentiation therapy as an effective clinical tool, furtherresearch is necessary to identify agents capable of efficiently inducingterminal differentiation in cancer cells without inducing nonspecifictoxicity in normal cells. Additionally, the identification of genes thatcorrelate with and may mediate terminal cell differentiation wouldrepresent valuable reagents for defining the molecular basis of terminalcell differentiation, for direct cancer therapeutic applications and forscreening compounds for potential use in differentiation therapy (6-12).

[0006] In cultured human melanoma cells, the combination of IFN-β+MEZresults in terminal cell differentiation and an irreversible loss ofproliferative potential (11,13,14). In this model system, a singletreatment for 24 h is sufficient to induce >95% terminal differentiationin cells subsequently grown for 72 h in the absence of inducers (14,15).The rapid induction of terminal differentiation in the vast majority oftreated cancer cells makes this system amenable for defining those geneexpression changes that occur during and that may mediate this process(11,12,16-19). To begin to address on a molecular level the question ofgrowth control and terminal differentiation in human melanoma cells andto directly clone genes involved in these processes we developed andused an efficient subtraction hybridization protocol (16). This approachhas resulted in the cloning of both known and novel cDNAs that aredifferentially regulated during growth suppression, reversibledifferentiation and terminal differentiation in human melanoma and othercancer cell types (16-20). The contents of U.S. Pat. No. 5,643,761,issued Jul. 1, 1997 to Fisher et al. entitled “Method for Generating aSubtracted cDNA Library and Uses of the Generated Library” and ofInternational Application PCT/US94/12160 filed Oct. 24, 1994, entitled“Method for Generating a Subtracted cDNA Library and Uses of theGenerated Library” which published May 4, 1995 as WO 95/11986 are herebyincorporated by reference.

[0007] In the present study, the properties of a novel mda-9 geneidentified by subtraction hybridization were described. mda-9 is anevolutionary conserved gene that encodes a protein of ˜32 to ˜34 kDawithout sequence homology to previously identified proteins. Expressionof mda-9 is seen in fifty human tissues, with slightly elevatedexpression in brain (putamen) and spleen (adult and fetal). Induction ofgrowth suppression and differentiation in human melanoma cells followingexposure to IFN-β+MEZ results in a decrease in mda-9 expression. Thesestudies provide additional support for the hypothesis that induction ofterminal differentiation and irreversible growth arrest in humanmelanoma cells involves multiple gene expression changes, includingincreases as well as decreases in the expression of specific targetgenes.

SUMMARY OF THE INVENTION

[0008] This invention provides a method for producing a temporallyspaced subtracted cDNA library comprising: a) isolating temporallyspaced RNAs from cells; b) generating cDNA inserts from the RNAsisolated from step (a); c) producing a temporally spaced cDNA libraryhaving clones containing the cDNA inserts generated from step (b); d)producing double stranded cDNA inserts from the temporally spaced cDNAlibrary; e) denaturing the double stranded cDNA inserts; f) contactingthe denatured double stranded cDNA inserts produced in step (e) withsingle-stranded DNAs from another cDNA library under conditionspermitting hybridization of the single-stranded DNAs and thedouble-stranded cDNA inserts; g) separating the hybridized cDNA insertsfrom the unhybridized inserts; h) generating a cDNA library of theunhybridized inserts, thereby generating a temporally spaced subtractedcDNA library.

[0009] This invention further provides a temporally spaced subtractedlibrary generated by the method for producing a temporally spacedsubtracted cDNA library comprising: a) isolating temporally spaced RNAsfrom cells; b) generating cDNA inserts from the RNAs isolated from step(a); c) producing a temporally spaced cDNA library having clonescontaining the cDNA inserts generated from step (b); d) producing doublestranded cDNA inserts from the temporally spaced cDNA library; e)denaturing the double stranded cDNA inserts; f) contacting the denatureddouble stranded cDNA inserts produced in step (e) with single-strandedDNAs from another cDNA library under conditions permitting hybridizationof the single-stranded DNAs and the double-stranded cDNA inserts; g)separating the hybridized cDNA inserts from the unhybridized inserts; h)generating a cDNA library of the unhybridized inserts, therebygenerating a temporally spaced subtracted cDNA library.

[0010] This invention provides a temporally spaced subtracted librarygenerated by using HO-1 melanoma cells treated with IFN-β and MEZ in atemporally spaced manner at 2, 4, 8, 12, 24, and 48 hours and, whereinthe single-stranded nucleic acid molecules are from another cDNA libraryof H0-1 melanoma cells.

[0011] This invention provides a method of identifying a melanomadifferentiation associated gene comprising: a) generating probes fromclones of the temporally spaced subtracted library generated by usingHO-1 melanoma cells treated with IFN-β and MEZ in a temporally spacedmanner at 2, 4, 8, 12, 24, and 48 hours and cells, wherein thesingle-stranded nucleic acid molecules are from another cDNA library ofH0-1 melanoma cells; and b) hybridizing the probe with the total RNAs ormRNAs from H0-1 cells treated with IFN-β and MEZ and the total RNAs ormRNAs from untreated H0-1 cells, hybridization of the probe with thetotal RNAs or mRNAs from the treated H0-1 cell but altered [no, reduced,or enhanced] hybridization with the total RNAs or mRNA from untreatedcells indicating that the clone from which the probe is generatedcarries a melanoma differentiation associated gene.

[0012] This invention provides a melanoma differentiation associatedgene identified by the above described method of identifying a melanomadifferentiation associated gene.

[0013] This invention provides a method of identifying temporallyexpressed genes from a single subtracted cDNA library, comprising: a)cloning the cDNAs from the temporally spaced subtracted cDNA libraryproduced by the above described method for producing a temporally spacedsubtracted cDNA library; b) hybridizing the clones obtained in step (a)with total RNAs isolated from control and with RNAs fromdifferentiation-inducer treated cells, hybridization of the probe RNAsfrom differentiation-inducer treated cells, either enhanced or no orreduced hybridization with total RNA isolated from control cellsindicating that the gene from which the probe was isolated is temporallyexpressed, thereby identifying temporally expressed genes from a singlesubtracted cDNA library.

[0014] This invention provides a temporally expressed gene identified bythe above described method.

[0015] This invention provides an isolated mda-9 gene. This inventionalso provides an isolated nucleic acid having the nucleic acid sequenceset forth in FIG. 7. This invention provides an isolated nucleic acidhaving the nucleic acid sequence set forth in FIG. 7, said nucleic acidencoding a human protein, wherein the encoded human protein is humanmda-9. This invention also provides a human mda-9 protein having theamino acid sequence set forth in FIG. 7.

[0016] This invention provides a method for identifying a compoundcapable of inducing terminal differentiation in cancer cells comprising:a) incubating an appropriate concentration of the cancer cells with anappropriate concentration of the compound; b) measuring the expressionof mda-9, the reduced expression of mda-9 gene indicating that thecompound is capable of inducing terminal differentiation in cancercells.

[0017] This invention provides a method for identifying a compoundcapable of inducing specific patterns of DNA damage caused by UVirradiation and gamma irradiation in human melanoma cells comprising: a)incubating an appropriate concentration of the human melanoma cells withan appropriate concentration of the compound; and b) measuring theexpression of mda-9, the altered expression of mda-9 gene indicatingthat the compound is capable of inducing specific patterns of DNA damagecaused by UV irradiation and gamma irradiation in human melanoma cells.

[0018] This invention provides a method for identifying a temporallyexpressed gene from cancer cells induced to undergo apoptosis by achemotherapeutic agent, comprising: a) incubating an appropriateconcentration of the cancer cells with an appropriate concentration ofthe chemotherapeutic agent; and b) measuring the expression of mda-9,the modified expression of mda-9 gene indicating that the compound iscapable of inducing the cancer cells to undergo apoptosis.

[0019] This invention provides a method for identifying a compoundcapable of elevating mda-9 expression in cancer cells comprising: a)incubating an appropriate concentration of the cancer cells with anappropriate concentration of the compound; b) measuring the expressionof mda-9 to determine whether the expression of the mda-9 gene iselevated.

[0020] This invention provides a method for detecting the presence ofcytokines in a sample comprising a) contacting the sample with cancerouscells; b) measuring the expression of the mda-9 gene; c) determiningwhether the expression of the mda-9 gene is altered, the alteredexpression of the mda-9 gene in the cancerous cells indicating thepresence of cytokines.

[0021] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to an mRNA moleculeencoding a human mda-9 protein so as to prevent expression of the mRNAmolecule. This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to an mRNA moleculeencoding a human mda-9 protein so as to prevent translation of the mRNAmolecule.

[0022] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to the promoter of theisolated nucleic acid molecule of an mda-9 gene, wherein the encodedmda-9 protein is a human protein, thereby preventing mRNA transcription.

[0023] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to the mRNA of the isolatednucleic acid molecule of an mda-9 gene, wherein the encoded mda-9protein is a human protein, and capable of degrading the hybridizedmRNA.

[0024] This invention provides a purified mda-9 protein. This inventionprovides a purified human mda-9 protein. This invention further providesa purified human mda-9 protein having an amino acid sequence as setforth in FIG. 7.

[0025] This invention provides an antibody directed to a purified mda-9protein. This invention provides an antibody directed to a purifiedhuman mda-9 protein. This invention further provides an antibodydirected to a purified human mda-9 protein having an amino acid sequenceas set forth in FIG. 7. This invention further provides an antibodycapable of specifically recognizing an mda-9 protein. In an embodimentof the invention, the antibody is capable of specifically recognizing ahuman mda-9 protein.

[0026] This invention provides a pharmaceutical composition comprisingan amount of the antisense oligonucleotide having a sequence capable ofspecifically hybridizing to an mRNA molecule encoding a human mda-9protein so as to prevent expression of the mRNA molecule, to preventtranslation of the mRNA molecule, effective to prevent expression of ahuman mda-9 protein and a pharmaceutically acceptable carrier. Thisinvention also provides a pharmaceutical composition comprising anamount of the antisense oligonucleotide having a sequence capable ofspecifically hybridizing to the promoter of the isolated nucleic acidmolecule of an mda-9 gene, wherein the encoded mda-9 protein is a humanprotein, thereby preventing mRNA transcription, effective to preventexpression of a human mda-9 protein and a pharmaceutically acceptablecarrier. This invention further provides a pharmaceutical compositioncomprising an amount of the antisense oligonucleotide having a sequencecapable of specifically hybridizing to the mRNA of the isolated nucleicacid molecule of an mda-9 gene, wherein the encoded mda-9 protein is ahuman protein and capable of degrading the hybridized mRNA, effective toprevent expression of a human mda-9 protein and a pharmaceuticallyacceptable carrier.

[0027] This invention provides a method of treating melanoma in asubject by administering a pharmaceutical composition comprising anamount of any one of the above described antisense oligonucleotideseffective to prevent expression of a human mda-9 protein and apharmaceutically acceptable carrier, thereby treating melanoma in asubject.

[0028] This invention provides a method of administering apharmaceutical composition comprising an amount of any one of the abovedescribed antisense oligonucleotides effective to prevent expression ofa human mda-9 protein and a pharmaceutically acceptable carrier.

[0029] This invention provides a method of inhibiting expression of amda-9 gene in a subject comprising introducing a vector containing anucleic acid molecule which renders the mda-9 gene functionless into thesubject under conditions permitting the inhibition of the expression ofthe mda-9 gene.

[0030] This invention provides a method of treating a cancer in asubject by administering a pharmaceutical composition comprising aneffective amount of the antibody capable of specifically recognizing anmda-9 protein, thereby treating the cancer in a subject.

[0031] This invention provides a method of increasing the expression ofmda-9 to inhibit cell growth comprising transfecting cells with anexpression vector comprising an mda-9 gene insert to induce expressionof mda-9 in cells thereby inhibiting growth of the cells.

[0032] This invention also provides a method of treating a cancer in asubject by increasing mda-9 expression in cancer cells of the subject toinduce partial differentiation in the cancer cells by administering apharmaceutical composition comprising a targeting vector and an agentwhich partially induces differentiation.

[0033] This invention further provides a method of treating a cancer ina subject by increasing mda-9 expression in cancer cells of the subjectto suppress growth of the cancer cells by administering a pharmaceuticalcomposition comprising a targeting vector and an agent which partiallyinduces differentiation.

[0034] This invention provides a cell having an exogenous indicator geneunder the control of the regulatory element of a mda-9 gene.

[0035] This invention provides a nucleic acid molecule comprising asequence of the promoter of an mda-9 gene protein.

BRIEF DESCRIPTION OF THE FIGURES

[0036]FIG. 1. Temporal expression of mda-9 in H0-1 cells exposed toIFN-β+MEZ. RNAs were isolated from untreated and H0-1 cells treated withIFN-β+MEZ (2000 U/ml+10 ng/ml) for 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h.Ten micrograms of total cellular RNA were separated on 1% agarose gel,transferred to nylon membranes, sequentially hybridized with an mda-9and then a GAPDH probe, and then exposed to autoradiography.

[0037]FIG. 2. Effects of IFN-β+MEZ on mda-9 expression in human melanomacells and an SV40-immortalized human melanocyte cell line. The indicatedcell line was grown in the presence or absence of IFN-β+MEZ (2000U/ml+10 ng/ml) for 96 h and RNA was analyzed as described in FIG. 1.

[0038]FIG. 3. Effect of interferons on growth of H0-1 cells. Cells weregrown for 96 h in the absence or presence of IFN-α (100 and 1000 U/ml),IFN-β (100 and 1000 U/ml) or IFN-γ (100 U/ml) and cell numbers intriplicate plates were determined. Results are the average cellnumber±S.D. from the mean.

[0039]FIG. 4. Effect of interferons on mda-9 expression in H0-1 cells.H0-1 cells were grown for 96 h in the absence or presence of IFN-α (100and 1000 U/ml), IFN-β (100 and 1000 U/ml) or IFN-γ (100 U/ml) and RNAwas analyzed as described in FIG. 1.

[0040]FIG. 5. Evolutionary conservation of the genomic mda-9 sequences.Genomic DNA (8 μg) isolated from different species, yeast (Saccharomycescerevisiae), cat, dog, monkey (Rhesus) and human (normal and HeLa), weredigested with HindIII. The digested DNAs were electrophoresed,transferred to nylon filters, hybridized with ³²P-labeled mda-9 geneprobe and exposed to autoradiography.

[0041]FIG. 6. Expression of mda-9 in human tissues. A positively chargednylon membrane containing poly A⁺ RNAs from the 50 tissues indicated washybridized with an mda-9 probe and exposed to autoradiography. The nylonmembrane was stripped, reprobed with a ubiquitin probe and exposed toautoradiography.

[0042]FIG. 7. Complete sequence of mda-9. Predicted translation of themda-9 cDNA begins at nucleotide 76 and ends at nucleotide 972. Accessionnumber AF006636 (Genbank). Mda-9 encodes a 298 amino acid protein withan M_(r) of 32,480.

[0043]FIG. 8. Effect of recombinant human gamma interferon (IFN-γ) onmda-9 expression in human melanocyte and melanoma cell lines. Theindicated cell typed were grown for 96 hr in the absence (C) or presenceof 100 U/ml of IFN-γ. Total RNA was isolated and analyzed by Northernblotting. Filters were probed with mda-9 then stripped and probed withGAPDH. The cell lines used include: H0-1 and C8161 from metastaticmelanomas; WM35 from a RGP (radial growth phase) primary melanoma; WM278from an early VGP (vertical growth phase) primary melanoma; and FM516-SVderived from a normal human melanocyte culture immortalized by the SV40T-antigen gene.

[0044] FIGS. 9A-9B. Temporal and dose kinetics of IFN-γ enhancement inmda-9 expression in H0-1 human melanoma cells. H0-1 cells were treatedfor the indicated time (FIG. 9A) and dose (FIG. 9B) of IFN-γ, RNA wasthen isolated and analyzed by Northern blotting. Results indicate thefold-change relative to control untreated cultures. Replicate samplesvaried by ≦15%.

DETAILED DESCRIPTION OF THE INVENTION

[0045] This invention provides a method for producing a temporallyspaced subtracted cDNA library comprising: a) isolating temporallyspaced RNAs from cells; b) generating cDNA inserts from the RNAsisolated from step (a); c) producing a temporally spaced cDNA libraryhaving clones containing the cDNA inserts generated from step (b); d)producing double stranded cDNA inserts from the temporally spaced cDNAlibrary; e) denaturing the double stranded cDNA inserts; f) contactingthe denatured double stranded cDNA inserts produced in step (e) withsingle-stranded DNAs from another cDNA library under conditionspermitting hybridization of the single-stranded DNAs and thedouble-stranded cDNA inserts; g) separating the hybridized cDNA insertsfrom the unhybridized inserts; h) generating a cDNA library of theunhybridized inserts, thereby generating a temporally spaced subtractedcDNA library.

[0046] As used herein “temporally spaced RNAs” are defined as RNAscollected over a sequential period of time. As used herein “temporallyspaced subtracted cDNA library” is a cDNA library generated using atemporally spaced cDNA library having clones containing the cDNA insertsgenerated from temporally spaced RNAs to which single-stranded DNAs fromanother cDNA library are hybridized and separated, resulting in the“subtracted” unhybridized cDNA insert library.

[0047] In an embodiment of the invention, the cDNA library used togenerate the single-stranded DNAs is from the same cell population asthe cell population used to generate the temporally spaced cDNA library.In a further embodiment of the invention, the cDNA library allowspropagation in single-stranded circle form. In a preferred embodiment ofthe invention, the cDNA library is a λZAP cDNA library.

[0048] In an embodiment of the invention, the double stranded cDNAinserts in step (d) are produced by releasing double-stranded cDNAinserts from double-stranded cDNA clones of the temporally spaced cDNAlibrary with appropriate restriction enzymes. In another embodiment ofthe invention, the single-stranded cDNAs are labeled with biotin. In anembodiment of the invention, the separating of step f) is performed byextraction with streptavidin-phenol: chloroform. In a preferredembodiment of the invention, the cells are HO-1 human melanoma cellstreated with IFN-β and MEZ. In a preferred embodiment of the invention,the treatment with IFN-β and MEZ is temporally spaced. In a furtherpreferred embodiment of the invention, the temporally spaced treatmentoccurs at 2, 4, 8, 12, 24, and 48 hours.

[0049] In an embodiment of the invention, the single-stranded nucleicacid molecules are from another cDNA library of H0-1 melanoma cells. Ina further embodiment of the invention, the cells are terminallydifferentiated and the single-stranded cDNAs are from another cDNAlibrary of undifferentiated cells. In another embodiment of theinvention, the cells are undifferentiated and the single-stranded cDNAsare from another cDNA library of terminally differentiated cells.

[0050] In a preferred embodiment of the invention, the cells arecancerous cells. In a further embodiment of the invention, the cancerouscells are selected from a group consisting of melanoma cells, basal cellcarcinoma cells, squamous cell carcinoma cells, neuroblastoma cells,glioblastoma multiforme cells, myeloid leukemic cells, breast carcinomacells, colon carcinoma cells, endometrial carcinoma cells, lungcarcinoma cells, ovarian carcinoma cells, prostate carcinoma cells,cervical carcinoma cells, osteosarcoma cells and lymphoma cells.

[0051] In an embodiment of the invention, the cells are induced toundergo reversible growth arrest, DNA damage, or apoptosis and thesingle-stranded cDNAs are from another cDNA library of uninduced cells.In another embodiment of the invention, the cells are uninduced cellsand the single-stranded cDNAs are from cells induced to undergoreversible growth arrest, DNA damage, or apoptosis.

[0052] As used herein, apoptosis is defined as programmed cell death.

[0053] In an embodiment of the invention, the cells are at onedevelopmental stage and the single-stranded cDNAs are from another cDNAlibrary of the cells at a different developmental stage. In anotherembodiment of the invention, the cells are cancerous and thesingle-stranded cDNAs are from another cDNA library from normal cells.In an embodiment of the invention, the cells are from the skin,connective tissue, muscle, breast, brain, meninges, spinal cord, colon,endometrium, lung, prostate and ovary.

[0054] This invention further provides a method further comprisingintroducing the subtracted library into host cells. In an embodiment ofthe invention, the method further comprises ligating the subtractedinserts into λ Uni-ZAP arms.

[0055] This invention further provides a temporally spaced subtractedlibrary generated by the method for producing a temporally spacedsubtracted cDNA library comprising: a) isolating temporally spaced RNAsfrom cells; b) generating cDNA inserts from the RNAs isolated from step(a); c) producing a temporally spaced cDNA library having clonescontaining the cDNA inserts generated from step (b); d) producing doublestranded cDNA inserts from the temporally spaced cDNA library; e)denaturing the double stranded cDNA inserts; f) contacting the denatureddouble stranded cDNA inserts produced in step (e) with single-strandedDNAs from another cDNA library under conditions permitting hybridizationof the single-stranded DNAs and the double-stranded cDNA inserts; g)separating the hybridized cDNA inserts from the unhybridized inserts; h)generating a cDNA library of the unhybridized inserts, therebygenerating a temporally spaced subtracted cDNA library.

[0056] This invention provides a temporally spaced subtracted librarygenerated by using HO-1 melanoma cells treated with IFN-β and MEZ in atemporally spaced manner at 2, 4, 8, 12, 24, and 48 hours and, whereinthe single-stranded nucleic acid molecules are from another cDNA libraryof H0-1 melanoma cells.

[0057] This invention provides a method of identifying a melanomadifferentiation associated gene comprising: a) generating probes fromclones of the temporally spaced subtracted library generated by usingHO-1 melanoma cells treated with IFN-β and MEZ in a temporally spacedmanner at 2, 4, 8, 12, 24, and 48 hours and cells, wherein thesingle-stranded nucleic acid molecules are from another cDNA library ofH0-1 melanoma cells; and b) hybridizing the probe with the total RNAs ormRNAs from H0-1 cells treated with IFN-β and MEZ and the total RNAs ormRNAs from untreated H0-1 cells, hybridization of the probe with thetotal RNAs or mRNAs from the treated H0-1 cell but altered [no, reduced,or enhanced] hybridization with the total RNAs or mRNA from untreatedcells indicating that the clone from which the probe is generatedcarries a melanoma differentiation associated gene. In an embodiment ofthe invention, the mRNAs are probed with labeled cDNA clones generatedfrom the temporally spaced subtracted library on a dot blot,hybridization of the probe with the mRNAs isolating a melanomadifferentiation associated gene.

[0058] This invention provides a melanoma differentiation associatedgene identified by the above described method of identifying a melanomadifferentiation associated gene.

[0059] This invention provides a method of identifying temporallyexpressed genes from a single subtracted cDNA library, comprising: a)cloning the cDNAs from the temporally spaced subtracted cDNA libraryproduced by the above described method for producing a temporally spacedsubtracted cDNA library; b) hybridizing the clones obtained in step (a)with total RNAs isolated from control and with RNAs fromdifferentiation-inducer treated cells, hybridization of the probe RNAsfrom differentiation-inducer treated cells, either enhanced or no orreduced hybridization with total RNA isolated from control cellsindicating that the gene from which the probe was isolated is temporallyexpressed, thereby identifying temporally expressed genes from a singlesubtracted cDNA library.

[0060] This invention provides a temporally expressed gene identified bythe above described method. In an embodiment of the invention, thetemporally expressed gene is cloned into a λ ZAP phage vector.

[0061] This invention provides an isolated mda-9 gene. In an embodimentof the invention, the isolated mda-9 gene is an isolated nucleic acid,wherein the encoded mda-9 protein is a human protein. This inventionalso provides an isolated nucleic acid having the nucleic acid sequenceset forth in FIG. 7. This invention provides an isolated nucleic acidhaving the nucleic acid sequence set forth in FIG. 7, said nucleic acidencoding a human protein, wherein the encoded human protein is humanmda-9. This invention also provides a human mda-9 protein having theamino acid sequence set forth in FIG. 7.

[0062] This invention provides a method for identifying a compoundcapable of inducing terminal differentiation in cancer cells comprising:a) incubating an appropriate concentration of the cancer cells with anappropriate concentration of the compound; b) measuring the expressionof mda-9, the reduced expression of mda-9 gene indicating that thecompound is capable of inducing terminal differentiation in cancercells. In an embodiment of the invention, the cancer cells are selectedfrom a group consisting of melanoma cells, basal cell carcinoma cells,squamous cell carcinoma cells, neuroblastoma cells, glioblastomamultiforme cells, myeloid leukemic cells, breast carcinoma cells, coloncarcinoma cells, endometrial carcinoma cells, lung carcinoma cells,ovarian carcinoma cells, prostate carcinoma cells, cervical carcinomacells, osteosarcoma cells and lymphoma cells.

[0063] This invention provides a method for identifying a compoundcapable of inducing specific patterns of DNA damage caused by UVirradiation and gamma irradiation in human melanoma cells comprising: a)incubating an appropriate concentration of the human melanoma cells withan appropriate concentration of the compound; and b) measuring theexpression of mda-9, the altered expression of mda-9 gene indicatingthat the compound is capable of inducing specific patterns of DNA damagecaused by UV irradiation and gamma irradiation in human melanoma cells.

[0064] This invention provides a method for identifying a temporallyexpressed gene from cancer cells induced to undergo apoptosis by achemotherapeutic agent, comprising: a) incubating an appropriateconcentration of the cancer cells with an appropriate concentration ofthe chemotherapeutic agent; and b) measuring the expression of mda-9,the modified expression of mda-9 gene indicating that the compound iscapable of inducing the cancer cells to undergo apoptosis. In anembodiment of the invention, the cancer cells are selected from a groupconsisting of melanoma cells, basal cell carcinoma cells, squamous cellcarcinoma cells, neuroblastoma cells, glioblastoma multiforme cells,myeloid leukemic cells, breast carcinoma cells, colon carcinoma cells,endometrial carcinoma cells, lung carcinoma cells, ovarian carcinomacells, prostate carcinoma cells, cervical carcinoma cells, osteosarcomacells and lymphoma cells.

[0065] This invention provides a method for identifying a compoundcapable of elevating mda-9 expression in cancer cells comprising: a)incubating an appropriate concentration of the cancer cells with anappropriate concentration of the compound; b) measuring the expressionof mda-9 to determine whether the expression of the mda-9 gene iselevated. In an embodiment of the invention, the compound capable ofelevating mda-9 expression in cancer cells is IFN-γ. In anotherembodiment of the invention, the compound capable of elevating mda-9expression in cancer cells is a cytokine. In a further embodiment of theinvention, the cytokine is selected from a group consisting of IFN-α,IFN-β, IFN-γ, TNF-α, stem cell growth factors, colony stimulatingfactor, GMCSF, and interleukins [including interleukin-6]. In a stillfurther embodiment of the invention, the cancer cells are selected froma group consisting of human melanoma cells, basal cell carcinoma cells,squamous cell carcinoma cells, neuroblastoma cells, glioblastomamultiforme cells, myeloid leukemic cells, breast carcinoma cells, coloncarcinoma cells, endometrial carcinoma cells, lung carcinoma cells,ovarian carcinoma cells, prostate carcinoma cells, cervical carcinomacells, osteosarcoma cells and lymphoma cells.

[0066] This invention provides a method for detecting the presence ofcytokines in a sample comprising a) contacting the sample with cancerouscells; b) measuring the expression of the mda-9 gene; c) determiningwhether the expression of the mda-9 gene is altered, the alteredexpression of the mda-9 gene in the cancerous cells indicating thepresence of cytokines.

[0067] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to an mRNA moleculeencoding a human mda-9 protein so as to prevent expression of the mRNAmolecule. This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to an mRNA moleculeencoding a human mda-9 protein so as to prevent translation of the mRNAmolecule.

[0068] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to the promoter of theisolated nucleic acid molecule of an mda-9 gene, wherein the encodedmda-9 protein is a human protein, thereby preventing mRNA transcription.

[0069] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to the mRNA of the isolatednucleic acid molecule of an mda-9 gene, wherein the encoded mda-9protein is a human protein, and capable of degrading the hybridizedmRNA.

[0070] This invention provides a purified mda-9 protein. This inventionalso provides a purified human mda-9 protein. This invention furtherprovides a purified human mda-9 protein having an amino acid sequence asset forth in FIG. 7.

[0071] This invention provides an antibody directed to a purified mda-9protein. This invention provides an antibody directed to a purifiedhuman mda-9 protein. This invention further provides an antibodydirected to a purified human mda-9 protein having an amino acid sequenceas set forth in FIG. 7. This invention further provides an antibodycapable of specifically recognizing an mda-9 protein. In an embodimentof the invention, the antibody is capable of specifically recognizing ahuman mda-9 protein. In an embodiment the antibody is capable ofspecifically recognizing a human mda-9 protein having an amino acidsequence as set forth in FIG. 7. In an embodiment of the invention, theantibody is a monoclonal or polyclonal antibody directed to a purifiedmda-9 protein. In another embodiment of the invention, the antibody is amonoclonal or polyclonal antibody capable of specifically recognizing anmda-9 protein. In another embodiment of the invention, the antibody is amonoclonal or polyclonal antibody capable of specifically recognizing ahuman mda-9 protein. In another embodiment of the invention, theantibody is a monoclonal or polyclonal antibody capable of specificallyrecognizing a human mda-9 protein having an amino acid sequence as setforth in FIG. 7. The above-described antibody is also useful for thedetection of mda-9 protein.

[0072] This invention provides a pharmaceutical composition comprisingan amount of the antisense oligonucleotide having a sequence capable ofspecifically hybridizing to an mRNA molecule encoding a human mda-9protein so as to prevent expression of the mRNA molecule, to preventtranslation of the mRNA molecule, effective to prevent expression of ahuman mda-9 protein and a pharmaceutically acceptable carrier. Thisinvention also provides a pharmaceutical composition comprising anamount of the antisense oligonucleotide having a sequence capable ofspecifically hybridizing to the promoter of the isolated nucleic acidmolecule of an mda-9 gene, wherein the encoded mda-9 protein is a humanprotein, thereby preventing mRNA transcription, effective to preventexpression of a human mda-9 protein and a pharmaceutically acceptablecarrier. This invention further provides a pharmaceutical compositioncomprising an amount of the antisense oligonucleotide having a sequencecapable of specifically hybridizing to the mRNA of the isolated nucleicacid molecule of an mda-9 gene, wherein the encoded mda-9 protein is ahuman protein and capable of degrading the hybridized mRNA, effective toprevent expression of a human mda-9 protein and a pharmaceuticallyacceptable carrier.

[0073] This invention provides a method of treating cancer in a subjectby administering the above described pharmaceutical compositioncomprising an amount of any one of the above described antisenseoligonucleotides effective to prevent expression of a human mda-9protein and a pharmaceutically acceptable carrier, thereby treatingmelanoma in a subject. In an embodiment, the cancer is selected from agroup consisting of human melanoma, basal cell carcinoma, squamous cellcarcinoma, neuroblastoma, glioblastoma multiforme carcinoma, myeloidleukemia, breast carcinoma, colon carcinoma, endometrial carcinoma, lungcarcinoma, ovarian carcinoma, prostate carcinoma, cervical carcinoma,osteosarcoma and lymphoma.

[0074] In an embodiment of the subject invention, the expression of ahuman mda-9 gene or protein is prevented by hybridization of anantisense oligonucleotide which is operatively linked to a tissuespecific promoter which is capable of directing the expression of theantisense oligonucleotide in the specific cancer cells. As used herein,“operatively linked” shall mean that the expression of the antisenseoligonucleotide is controlled by the tissue specific promoter. Inanother embodiment, the expression of a human mda-9 protein is preventedby hybridization of the antisense oligonucleotide to the mda-9 genepromoter or mda-9 mRNA molecules regulated by a tissue specific promoterthat permits expression of the human mda-9 antisense sequence only inmelanocyte and melanoma cells. In a further embodiment, the cancer ismelanoma and the tissue specific promoter is a tyrosinase promoter.

[0075] This invention provides a method of administering apharmaceutical composition comprising an amount of any one of the abovedescribed antisense oligonucleotides effective to prevent expression ofa human mda-9 protein and a pharmaceutically acceptable carrier. In anembodiment of the invention, pharmaceutical composition furthercomprises a substance which facilitates the delivery of saidoligonucleotide into the cell. As used herein, the substance whichfacilitates the delivery of the oligonucleotide into the cell may be aliposome or an antibody. In an embodiment of the invention, theoligonucleotide is introduced into the cell by a viral vector. In anembodiment of the invention, the oligonucleotide is stabilized, so asnot to be degraded.

[0076] This invention provides a method of inhibiting expression of amda-9 gene in a subject comprising introducing a vector containing anucleic acid molecule which renders the mda-9 gene functionless into thesubject under conditions permitting the inhibition of the expression ofthe mda-9 gene.

[0077] As used herein, “functionless” is defined as inability of themda-9 gene to encode the mda-9 protein, including inability totranscribe the mda-9 gene, or inability to translate the mda-9 protein.

[0078] In an embodiment of the invention, the nucleic acid is anantisense oligonucleotide having a sequence capable of specificallyhybridizing to an mRNA molecule encoding a human mda-9 protein. Inanother embodiment of the invention, the nucleic acid contains amutation or deletion of the mda-9 gene having the appropriate flankingsequences.

[0079] As used herein, the appropriate flanking sequences are defined asthe sequences required in order for recombination to occur.

[0080] This invention provides a method of treating a cancer in asubject by administering a pharmaceutical composition comprising aneffective amount of the antibody capable of specifically recognizing anmda-9 protein, thereby treating the cancer in a subject. In anembodiment of the invention, the cancer is a melanoma.

[0081] This invention provides a method of increasing the expression ofmda-9 to inhibit cell growth comprising transfecting cells with anexpression vector comprising an mda-9 gene insert to induce expressionof mda-9 in cells thereby inhibiting growth of the cells. In anembodiment of the invention, the mda-9 gene insert is in either thesense or antisense orientation. In another embodiment of the invention,the mda-9 gene insert is in the sense orientation and mda-9 isoverexpressed. In an embodiment of the invention, the mda-9 gene insertis in the antisense orientation and mda-9 expression is inhibited. Inanother embodiment the cells are selected from the group consisting ofhuman melanoma, basal cell carcinoma, squamous cell carcinoma,neuroblastoma, glioblastoma multiforme carcinoma, myeloid leukemia,breast carcinoma, colon carcinoma, endometrial carcinoma, lungcarcinoma, ovarian carcinoma, prostate carcinoma, cervical carcinoma,osteosarcoma and lymphoma.

[0082] This invention provides a method of treating a cancer in asubject by increasing mda-9 expression in cancer cells of the subject toinduce partial differentiation in the cancer cells by administering apharmaceutical composition comprising a targeting vector and an agentwhich partially induces differentiation. In an embodiment of theinvention, the targeting vector is an mda-9 expression vector and saidvector is selected from the group consisting of an adenovirus, anadeno-associated virus, a retrovirus, a vaccinia virus, and an EpsteinBarr virus. In another embodiment of the invention, the agent whichpartially induces differentiation is a cytokine, a DNA damagingchemotherapeutic agent, or a physical therapeutic agent. In anembodiment of the invention, the cytokine is selected from the groupconsisting of IFN-α, IFN-β, IFN-γ and TNF-α. In another embodiment ofthe invention, the DNA damaging chemotherapeutic agent is selected fromthe group consisting of dactinomycin, cis-platinum, and taxol and itsanalogs. In an embodiment of the invention, the physical therapeuticagent is gamma irradiation. In an embodiment of the invention, thecancer cells are selected from the group consisting of human melanoma,basal cell carcinoma, squamous cell carcinoma, neuroblastoma,glioblastoma multiforme carcinoma, myeloid leukemia, breast carcinoma,colon carcinoma, endometrial carcinoma, lung carcinoma, ovariancarcinoma, prostate carcinoma, cervical carcinoma, osteosarcoma andlymphoma.

[0083] This invention provides a method of treating a cancer in asubject by increasing mda-9 expression in cancer cells of the subject tosuppress growth of the cancer cells by administering a pharmaceuticalcomposition comprising a targeting vector and an agent which partiallyinduces differentiation. In an embodiment of the invention, thetargeting vector is an mda-9 expression vector and said vector isselected from the group consisting of an adenovirus, an adeno-associatedvirus, a retrovirus, a vaccinia virus, and an Epstein Barr virus. Inanother embodiment of the invention, the agent which partially inducesdifferentiation is a cytokine, a DNA damaging chemotherapeutic agent, ora physical therapeutic agent. In an embodiment of the invention, thecytokine is selected from the group consisting of IFN-α, IFN-β, IFN-γand TNF-α. In another embodiment of the invention, the DNA damagingchemotherapeutic agent is selected from the group consisting ofdactinomycin, cis-platinum, and taxol and its analogs. In an embodimentof the invention, the physical therapeutic agent is gamma irradiation.In an embodiment of the invention, the cancer cells are selected fromthe group consisting of human melanoma, basal cell carcinoma, squamouscell carcinoma, neuroblastoma, glioblastoma multiforme carcinoma,myeloid leukemia, breast carcinoma, colon carcinoma, endometrialcarcinoma, lung carcinoma, ovarian carcinoma, prostate carcinoma,cervical carcinoma, osteosarcoma and lymphoma.

[0084] This invention provides a cell having an exogenous indicator geneunder the control of the regulatory element of a mda-9 gene. In anembodiment of the invention, the cell is a normal cell. In anotherembodiment of the invention, the cell is a cancer cell. In any of theabove-described embodiments of a cell having an exogenous indicator geneunder the control of the regulatory element of a mda-9 gene, theindicator gene may code for beta-galactosidase, luciferase,chloramphenicol transferase or secreted alkaline phosphatase.

[0085] This invention provides a method for determining whether an agentis capable of modifying DNA damage and repair pathways, differentiation,apoptosis or operates through a cytokine modulatory pathway comprisingcontacting an amount of the agent with the cell of claim 88, wherein achange in expression of the indicator gene compared to the expression incontrol cells indicates that the agent modifies DNA damage and repairpathways, differentiation, apoptosis or operates through a cytokinemodulatory pathway. The ability of an agent to modify DNA damage, modifyDNA repair pathways, modify cell differentiation, modify apoptosis or tooperate through a cytokine modulatory pathway may be mutually exclusiveor an agent may also be capable of more than one type of activity ormodification. In an embodiment of the invention, the change inexpression is either a decrease in expression or an increase inexpression of the indicator gene.

[0086] One of skill in the art will select appropriate control cells forthe screening methods depending upon the desired “end point”characteristics sought for the screened molecules or agents. Controlcells may be selected from, but not limited to, cells treated with asolvent rather than the molecule or the agent performing theabove-described modifications, an agent which does not induce DNAdamage, or a reporter-promoter construct driven by a constitutivepromoter such as a CMV (cytomegalovirus) promoter. A desired smallmolecule or agent characteristic e.g. “end point” which is selected forby the screening method may be selected from, but not limited to, achange in cellular morphology, growth suppression of cells, upregulationof mda-9 or modification of any specific gene.

[0087] This invention provides a nucleic acid molecule comprising asequence of the promoter of an mda-9 gene protein.

[0088] This invention provides a method to screen for either a smallmolecule or agent which modifies DNA damage and repair pathways,differentiation, or apoptosis, or operates through a cytokine modulatorypathway comprising: a) contacting an amount of the small molecule oragent with cells of claim 88; b) determining expression of the indicatorgene; c) comparing the expression determined in step (b) with controlcells, a modified expression of the indicator gene in the cells of step(a) compared to the control cells indicating that the small molecule oragent modifies DNA damage and repair pathways, differentiation,apoptosis or operates through a cytokine modulatory pathway.

[0089] In an embodiment of the invention, the modification in expressionis either a decrease in expression or an increase in expression of theindicator gene. In another embodiment of the invention, the smallmolecule which alters the expression of the mda-9 gene or DNA-damagingagent is selected from a recombinatorial library, a peptide library, apeptide-derived library or a chemical library or a combinatorialchemical library. “Chemical libraries” have been defined as“intentionally created collections of differing molecules which can beprepared synthetically or biosynthetically”. A type of syntheticstrategy which can lead to large chemical libraries is “combinatorialchemistry”. “Combinatorial chemistry” has been defined as “thesystematic and repetitive, covalent connection of a set of different‘building blocks’ of varying structures to each other to yield a largearray of diverse molecular entities.” (Gallop, M. A. et al. (1994) J. ofMed. Chem. 37(9) 1233-1251.) Building blocks can include nucleotides,carbohydrates, peptides or peptoids into ordered structures. Chemicallibraries generated utilizing combinatorial chemistry can displayremarkable diversity. Peptide libraries may be selected from peptides orpeptide mimics. The contents of WO 95/20591 (Stolowitz, M.) Aug. 3,1995; WO/96/04403 (Burke, D. et al.) Feb. 15, 1996; WO 96/09316 (Eatonand Gold) Mar. 28, 1996; WO 96/27605(Gold, L. et al.) Sep. 12, 1996which disclose inter alia production of chemical libraries,combinatorial libraries, and nucleotide libraries, are herebyincorporated by reference.

[0090] This invention provides an agent which is a small moleculeselected from a recombinatorial library, a peptide library, apeptide-derived library or a chemical library. One of skill in the artwill know which type of library is required for the screening methoddepending upon the desired characteristics of molecules to be screenedfor. Recombinatorial libraries may be chemically synthesized, e.g. achange in one atom of a chemical compound would create numerous“unknown” chemicals to be screened for a desired characteristic. Thescreening method would enable one of skill in the art to screen forsmall molecules or agents having the same properties or characteristicsas an entire molecule, e.g. a small molecule with properties of a wholwmolecule, e.g. having properties of a cytokine. The above describedscreening methods, e.g. using a reporter-promoter construct as acontrol, would enable one of skill in the art to find therapeutic agentswhich alter specific genes.

[0091] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

[0092] First Series of Experiments

[0093] Experimental Details

[0094] Cell Lines and Culture Conditions. H0-1 is a melanotic melanomacell line produced from a metastatic inguinal lymph node lesion from a49 year-old female and was used between passages 150 and 175 (13,21,22).FMS16-SV is a normal human melanocyte culture immortalized by the SV40T-antigen gene (23). Additional melanoma cell lines established frompatients with metastatic melanomas that were evaluated, include LO-1,SH-1, WM239, MeWo, SKMEL-p53 wt (containing a wild-type p53 gene) andSKMEL-p53 mut (containing a mutant p53 gene) (13,21,24). Cultures weregrown at 37° C. in a 95% air 5% C0₂-humidified incubator in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 5 or 10% fetal bovineserum (Hyclone, Utah). Cultures were maintained in the logarithmic phaseof growth by subculturing (1:5 or 1:10) prior to confluenceapproximately every 4 to 7 d. For determining steady-state RNAexpression, cultures were seeded at 1.5×10⁶ cells per 10-cm tissueculture plate, and 24 h later the medium was changed without inducers orwith IFN-β (2000 U/ml), MEZ (10 ng/ml) or IFN-β+MEZ (2000 U/ml+10ng/ml). Total cytoplasmic RNA was isolated at various time points andanalyzed for mda-9 and GAPDH expression.

[0095] Cloning of mda-9 by Subtraction Hybridization. Identification andcloning of mda-9 was accomplished as described previously (16). Briefly,a cDNA library was prepared from RNA isolated from actively growing H0-1cells (driver) and RNAs isolated from H0-1 cells treated with IFN-β+MEZ(2000 U/ml+10 ng/ml) for 2, 4, 8, 12 and 24 h (temporally spacedtester). Subtraction hybridization was then performed betweendouble-stranded tester DNA and single-stranded driver DNA prepared bymass excision of the libraries. The TSS cDNAs were efficiently clonedinto the λ Uni-ZAP phage vector and used to screen Northern blotscontaining total RNA isolated from control H0-1 cells and H0-1 culturestreated for 24 h with IFN-β (2000 U/ml), MEZ (10 ng/ml) or IFN-β+MEZ(2000 U/ml+10 ng/ml). This strategy resulted in the identification of apartial mda-9 cDNA (11). A full-length mda-9 cDNA was isolated followingscreening of a differentiation inducer-treated H0-1 cDNA library (16)and using the procedure of rapid amplification of cDNA ends (RACE) asdescribed previously (17,25-27). Sequence analysis was determined asdescribed (28,29).

[0096] RNA Isolation, Northern Blotting and Southern Blotting of GenomicDNAs. Total cellular RNA was isolated by the guanidinium/phenolprocedure and Northern blotting was performed as described (30-32). Tenμg of RNA were denatured with glyoxal/dimethyl sulfoxide (DMSO),electrophoresed on 1.0% agarose gels, transferred to nylon membranes andhybridized to a ³²P-labeled mda-9 probe and then after stripping themembranes were hybridized to a ³²P-labeled rat GAPDH probe (33) asdescribed previously (30-32). Following hybridization, the filters werewashed and exposed for autoradiography. RNA blots were quantitated bydensitometric analysis using a Molecular Dynamics densitometer(Sunnyvale, Calif.) (34). To determine human tissue specific expressionof mda-9 a Human RNA Master Blot™ (Clontech Laboratories, Inc., PaloAlto, Calif.), containing poly A⁺ RNA from 50 tissues immobilized asseparate dots on a charged nylon membrane, was probed with a ³²P-labeledmda-9 cDNA probe and following stripping the membrane was probed with a³²P-labeled human ubiquitin housekeeping cDNA probe as described byClontech Laboratories, Inc. Following hybridization, the filters werewashed and exposed for autoradiography.

[0097] Genomic DNAs for Saccharomyces cerevisiae (yeast), cat, dog,Rhesus monkey and normal human were obtained commercially (PromegaCorp., Madison, Wis. and Clontech Laboratories Inc., Palo Alto, Calif.).Human DNA was also prepared from HeLa human cervical carcinoma cells.The DNAs were digested completely with HindIII restriction enzyme,electrophoresed, transferred to nylon membranes and hybridized to a³²P-labeled mda-9 gene probe (16,18,32). After hybridization the nylonmembranes were washed in 3×SSC, 0.1 SDS, 30 min; 1×SSC, 0.1 SDS, 30 min;and 0.1×SSC, 0.1% SDS, 20 min at 55° C.; and then exposed toautoradiography (18).

[0098] Reagents. Recombinant human IFN-β, with a serine substituted fora cysteine at position 17 of the molecule (35), was provided by TritonBioscience (Alameda, Calif.). IFN-β was obtained as a lyophilized powderwith a concentration of 4.5×10 ⁷ U/ml. Recombinant human IFN-α (IFN-αA)was provided by Hoffmann-La Roche, Inc., NJ. Recombinant human IFN-γ waskindly provided by Dr. Sidney Pestka (UMDNJ-Robert Wood Johnson MedicalSchool, NJ). The interferon titer of IFN-αA and IFN-β was determinedusing a cytopathic effect inhibition assay with vesicular stomatitisvirus (VSV) on a bovine kidney cell line (MDBK) or human fibroblastAG-1732 cells (36). The interferon titer of IFN-γ was determined using acytopathic effect inhibition assay with VSV on the human WISH cell line(36). The concentrated stocks of interferons were diluted to 1×10⁶ U/mlin DMEM-10, frozen at −80° C., thawed immediately prior to use, anddiluted to the appropriate concentration in DMEM-10. Stock solutionswere maintained at 4° C. MEZ was obtained from Sigma Scientific Co. (St.Louis, Mo.). Stock solutions were prepared in DMSO, aliquoted into smallportions, and stored at −20° C. The final concentration of DMSO did notalter growth or induce markers of differentiation (elevated melaninsynthesis) in the cell lines used in the present study.

[0099] Experimental Results

[0100] mda-9 Is Variably Expressed in H0-1 Cells Treated with IFN-β+MEZ.The subtraction hybridization strategy employed to identify genesinvolved in terminal cell differentiation has a high probability ofdetecting genes that display elevated expression in IFN-β+MEZ treatedversus actively proliferating control H0-1 human melanoma cells (11).However, since cDNA libraries were constructed from pooled RNA samplesobtained from H0-1 cells treated for various times with IFN-β+MEZ, i.e.,2, 4, 8, 12 and 24 h, it is equally possible that genes displayingbiphasic patterns of gene expression can also be isolated from this TSScDNA library. This is indeed the case as found with mda-9 which displaysmaximum enhanced expression 8 and 12 h after treatment with IFN-β+MEZ,whereas expression is lower than controls after 1 or 24 h treatment(FIG. 1). Exposure to IFN-β+MEZ for 2 or 4 h also elevates mda-9expression, but to a lesser extent than after 8 or 12 h. On the basis ofthis study, if subtracted cDNA libraries had been produced solely fromH0-1 cells treated for 24 h with IFN-β+MEZ the probability of isolatingmda-9 cDNA clones would be significantly reduced. In this context, thetemporally spaced subtracted (TSS) IFN-β+MEZ cDNA library should permitthe cloning of additional genes that only display elevated expression inhuman melanoma cells during specific times within the first 24 h oftreatment with IFN-β+MEZ.

[0101] mda-9 Is Down-Regulated in Terminally Differentiated HumanMelanoma Cells. Treatment of human melanoma cells with IFN-β+MEZ (2000U/ml+10 ng/ml) for 96 h results in growth suppression and terminal celldifferentiation in the majority of treated cells (13,14,17). When grownin the single agent, growth suppression is less and the degree ofinhibition depends on the specific melanoma analyzed (Table 1).Moreover, cultures treated with a single agent are not terminallydifferentiated (data not shown). The combination of agents eithersynergistically or additively reduces growth, depending on the melanomacell line studied. Growth suppression induced by the combination ofagents in specific melanomas is independent of the in vitro growth rateof these cells. For example, a >90% inhibition in growth is seenfollowing 96 h treatment with IFN-β+MEZ in slow growing human melanomas,such as L0-1 and SKMEL-p53 wt, as well as rapidly growing humanmelanomas, such as H0-1 and WM239. Maximum growth suppression, >95% incomparison with untreated control cultures, is apparent in humanmelanoma cells, H0-1, L0-1 and SKMEL-p53 wt, encoding a wild-type p53protein (Table 1). Two human melanoma cells with a previously definedmutation in p53, MeWo and SKMEL-p53 mut, display <75% reduction ingrowth when treated with IFN-β+MEZ. In contrast, WM239, with animmunologically mut p53 protein, displays a different profile ofsensitivity than the other melanoma cells. WM239 cells treated withIFN-β+MEZ are inhibited by ˜91% when grown in the combination of agents(Table 1). Growth of the SV40-immortalized human melanocyte cell line,FM516-SV, in IFN-β+MEZ results in an ˜70% reduction in growth withoutinducing terminal differentiation in the majority of treated cells (17)(Table 1 and data not shown).

[0102] To determine if induction of differentiation modifies mda-9expression, RNAs were isolated from human melanoma and FM516-SV cellsgrown for 96 hr in the absence or presence of IFN-β+MEZ (2000 U/ml+10ng/ml) (FIG. 2). In all of the cell lines, the combination of IFN-β+MEZdecreases the steady-state level of mda-9 RNA. Based on densitometercomparisons of mda-9 and GAPDH RNA levels, mda-9 RNA expression isreduced from ˜1.5- to ˜14-fold in treated cultures, with MeWo cellsshowing the greatest change and FM516-SV cells displaying the smallestchange. A direct relationship between the level of reduction in mda-9expression and the degree of growth suppression induced by IFN-β+MEZ isnot apparent in the melanoma cell lines used in the present study. TABLE1 Effect of IFN-β and MEZ, alone and in combination, on the growth ofhuman melanoma and melanocyte cell lines Experimental Conditions^(a)Cell Line Control IFN-β MEZ IFN-β + MEZ HO-1 64.5 ± 5.3 14.0 ± 2.6 16.8± 1.0 (26) 0.7 ± 0.2 (1) (22) LO-1 17.0 ± 2.0  1.3 ± 0.3  4.7 ± 0.4 (28)0.4 ± 0.1 (2) (8) MeWo 33.6 ± 1.2 15.6 ± 1.9 15.4 ± 1.9 (46) 9.4 ± 1.1(28) (46) SH-1 24.9 ± 1.6 14.3 ± 1.5 14.1 ± 0.3 (57) 8.0 ± 1.2 (32) (57)SKMEL-p53 36.1 ± 4.8 39.5 ± 7.5 21.5 ± 4.3 (60) 9.3 ± 0.6 (26) mut (109)SKMEL-p53 16.8 ± 0.8  9.1 ± 0.3  3.6 ± 0.7 (21) 0.7 ± 0.2 (4) wt (54)WM239 49.8 ± 1.8  9.5 ± 0.9 19.7 ± 1.2 (40) 4.4 ± 0.4 (9) (19) FM516-SV21.2 ± 1.7  7.8 ± 1.0 12.7 ± 1.8 (60) 6.2 ± 0.6 (29) (37)

[0103] Treatment of H0-1 Cells with Interferon Induces GrowthSuppression While Enhancing mda-9 Expression. Experiments were performedto determine if a relationship exists between growth suppression andreduced mda-9 expression. To test this connection, the effect ofinterferon and MEZ treatment on proliferation and mda-9 expression inhuman melanoma cells was determined. Growth of H0-1 cells in thepresence of type I interferon, IFN-α or IFN-β, or type II interferon,IFN-γ, suppresses the growth of H0-1 cells (FIG. 3). IFN-β is the mosteffective interferon in inhibiting H0-1 growth, with an ˜70% reductionafter 4 d treatment with 100 U/ml. Under similar experimentalconditions, 100 U/ml of IFN-γ reduces growth by ˜43% and 100 U/ml ofIFN-A reduces growth by only ˜23%. Unlike IFN-β+MEZ, which reduce mda-9expression in H0-1 cells, all three interferons enhance mda-9 expression˜1.9- to ˜4.0-fold based on equalization for GAPDH expression (FIG. 4).In contrast, mda-9 expression is unaffected in H0-1 cells grown for 4days in 10 ng/ml of MEZ, even though growth is reduced by ˜74% (Table 1and data not shown). These results indicate that growth suppression inH0-1 cells can be dissociated from decreased mda-9 expression.

[0104] mda-9 Is an Evolutionary Conserved Gene. To determine ifsequences homologous to human mda-9 are present in the genomes of otherspecies Southern blotting analyses were performed using genomic DNAsfrom Saccharomyces cerevisiae (yeast), cat, dog, Rhesus monkey and human(normal and HeLa) (18) (FIG. 5). On the basis of intensity ofhybridization in Southern blots, the greatest sequence homology occursbetween monkey and human genomic DNAs. Hybridization with yeast DNA isalso evident. The apparently high level of hybridization with the mda-9probe is the result of an ˜10-fold higher relative concentration ofgenomic yeast DNA added to this gel. Dog and cat genomic DNA displayweaker hybridization after probing Southern blots with mda-9. Thesefindings suggest that mda-9 is an evolutionary conserved gene.

[0105] mda-9 Is Expressed in Diverse Human Tissues. To determine thepattern of expression of the mda-9 gene a Human RNA Master Blot™ thatcontains poly A⁺ RNAs from 50 human tissues immobilized in separate dotson a nylon membrane was analyzed (FIG. 6). As a positive control for RNAexpression the membranes were stripped and rehybridized with a ubiquitincDNA probe (FIG. 6). Both mda-9 and ubiquitin are expressed in all 50human tissues. Comparing the intensity of hybridization between mda-9and ubiquitin, elevated expression of mda-9 occurs in putamen, adultspleen and fetal spleen and reduced expression occurs in whole brain,amygdala, caudate nucleus, cerebellum, cerebral cortex, frontal lobe,hippocampus, medulla oblongata, occipital lobe, substantia nigra,temporal lobe, thalamus, sub-thalamic nucleus, spinal cord, heart,aorta, skeletal muscle, colon, bladder, uterus, prostate, stomach,testis, ovary, pancreas, pituitary gland, adrenal gland, thyroid gland,salivary gland, mammary gland, kidney, liver, small intestine, thymus,peripheral blood leukocytes, lymph node, bone marrow, appendix, lung,trachea and placenta. mda-9 and ubiquitin are also expressed in fetalbrain, fetal heart, fetal kidney, fetal liver, fetal thymus and fetallung. Hybridization with mda-9 also occurs with Escherichia coli DNA andwith human genomic DNA (500 ng) (FIG. 6). Longer exposures of the HumanMaster RNA Blot™ probed with ubiquitin indicates hybridization to humangenomic DNA (both 100 and 500 ng), but no hybridization with Escherichiacoli DNA (data not shown). No hybridization is observed with mda-9 orubiquitin with yeast total RNA, yeast tRNA, Escherichia coli rRNA orpoly r(A). The ability of mda-9 to hybridize to Escherichia colisuggests some homology to bacterial sequences. DNA data bank sequencesearches indicate only minor homology within small regions of mda-9 andbacterial sequences. The ability of mda-9 to hybridize with humangenomic DNA may indicate the presence of repetitive sequences or thatthis gene is highly abundant or a member of a multi-gene family.Analysis of multiple human tissue Northern blots also demonstratesfairly uniform mda-9 expression in multiple tissue types (data notshown).

[0106] Experimental Discussion

[0107] The aberrant differentiation/modified gene expression model ofcancer development is based on the hypothesis that specific forms ofcancer may develop from defects in differentiation and gene expressionthat are inherently reversible (6-11,13,14). If these assumptions arecorrect, then it may be possible to induce the appropriate program ofgene expression and a more normal differentiated phenotype in a cancercell by treatment with the appropriate agent(s). This idea has beenexperimentally tested using cultured human melanoma cells (13,14,16).The combination of IFN-β+MEZ results in changes in the expression of aspectrum of genes, including cell cycle and growth regulating genes, andthe induction of an irreversible loss in proliferative ability andterminal differentiation in malignant melanoma cells (13-18,34). Byusing the molecular approach of subtraction hybridization those changesin gene expression that correlate with and may control growth anddifferentiation in human cancer cells are being defined (16-20). Thisinformation offers potential for identifying potentially new cellulartargets for the differentiation therapy of human cancer.

[0108] In the present study a novel mda-9 gene identified by subtractionhybridization that is down-regulated when human melanoma cells areinduced to terminally differentiate is described. Decreased expressionof mda-9 in H0-1 cells can be distinguished from growth suppression orinduction of specific markers of differentiation, such as enhancedmelanin synthesis and the formation of dendrite-like processes. Forexample, agents that suppress growth in H0-1 cells without inducingmarkers of differentiation, such as IFN-γ, elevate mda-9 expression,whereas MEZ, which can reversibly induce elevated melanin synthesis,growth suppression and dendrite-like processes in H0-1 cells, has noeffect on mda-9 expression. These findings suggest that mda-9 may be acomponent of the terminal differentiation program in human melanomacells. Southern blotting using genomic DNAs from different speciesindicates that mda-9 is an evolutionary conserved gene and analysis ofmultiple human tissue-derived mRNAs indicate that mda-9 is a widelyexpressed gene, with a small elevation in expression in the putamen(brain) and spleen (both adult and fetal). Nucleotide and amino acidsequence analysis of mda-9 indicate no significant homology topreviously reported genes. However, two small stretches of homology, 22of 69 (31%) and 11 of 45 (24%) identities and 36 of 69 (52%) and 22 of29 (75%) positives, respectively, are apparent between mda-9 (aa 196 to264 and aa 135 to 179) and the X11 gene product (aa 637 to 705 and 554to 598) (37). The X11 gene encodes a protein that is expressed in thebrain, primarily in the granular layer of the cerebellum, but is notdetectable in several non-neuronal tissues and cell lines. The X11 geneencodes a protein of 708-aa with a putative transmembrane segment andmay represent a candidate Friedreich ataxia gene (37). Since thehomologies between mda-9 and X11 are so small, it is unlikely that thesegenes display functional similarities.

[0109] Further studies are required to determine the biologicalrelevance of modified mda-9 expression in terminal differentiation inhuman melanoma cells. For example, overexpression of mda-9 in melanomacells could be used to determine if preventing down-regulation of mda-9can modify the differentiation process. Alternatively, inhibiting mda-9expression by using antisense based technologies can also be used toevaluate the role of this gene in terminal differentiation in humanmelanoma cells. Additional studies are also required to determine ifaltered mda-9 expression is associated with the differentiation orgrowth processes in other cancer and normal cell types. Experiments todetermine the spectrum of cytokine and differentiation-modulating agentsthat can affect mda-9 expression will also be informative.

[0110] The gene expression changes associated with and mediatingterminal differentiation in human melanoma cells are complex, consistingof both increases and decreases in the abundance of specific RNA species(10-12,14-20,34). Unraveling the roles of those gene products thatpositively regulate differentiation phenotypes and that negativelyregulate growth is essential in order to define terminal differentiationon a molecular level. Several models are possible for integrating thesegene changes in the terminal differentiation process. A ‘master-switch’gene may exist that can singularly induce the cascade of gene expressionchanges resulting in terminal differentiation, i.e., differentiation isa linear process initially controlled by a single genetic element.Treatment of cells with an agent(s) that induces terminaldifferentiation may result in the induction and suppression of parallelsets of genes that ultimately converge to induce terminaldifferentiation, i.e., differentiation involves multiple independentpathways resulting in the activation of common genes mediating terminaldifferentiation. Alternatively, several independent and overlappingpathways may control differentiation, i.e., differentiation involvesfeedback loops consisting of multiple genes that display either elevatedor decreased expression and that control the expression of downstreamgenes and pathways critical for differentiation. Further studies shouldhelp clarify the molecular and biochemical processes that control theinduction and maintenance of terminal differentiation in human melanomacells and delineate the roles of specific mda genes in regulating theseprocesses.

REFERENCES FOR FIRST SERIES OF EXPERIMENTS

[0111] 1. Fisher, P. B., Enhancement of viral transformation andexpression of the transformed phenotype by tumor promoters. In: T. J.Slaga, Ed., Tumor Promotion and Cocarcinogenesis In Vitro, Mechanisms ofTumor Promotion, pp. 57-123, CRC Press, Boca Raton, Fla., 1984.

[0112] 2. Bishop, J. M., Molecular themes in oncogenesis. Cell, 64:235-248, 1991.

[0113] 3. Vogelstein, B., and Kinzler, K. W., The multistep nature ofcancer. Trends Genet., 9: 138-141, 1991.

[0114] 4. Knudson, A. G., Antioncogenes and human cancer. Proc. Natl.Acad. Sci. U.S.A., 90: 10914-10921, 1993.

[0115] 5. Hartwell, L. H., and Kastan, M. B., Cell cycle control andcancer. Science, 266: 1821-1828, 1994.

[0116] 6. Waxman, S., Rossi, G. B., and Takaku, F., Eds., The Status ofDifferentiation Therapy, Vol. 1, Raven Press, New York, 1988.

[0117] 7. Fisher, P. B., and Rowley, P. T., Regulation of growth,differentiation and antigen expression in human tumor cells byrecombinant cytokines: applications for the differentiation therapy ofcancer. In: Waxman, S., Rossi, G. B., and Takaku, F., Eds., The Statusof Differentiation Therapy of Cancer, Vol. 2, pp. 201-213, Raven Press,New York, 1991.

[0118] 8. Waxman, S., Rossi, G. B., and Takaku, F., Eds., The Status ofDifferentiation Therapy, Vol. 2, Raven Press, New York, 1991.

[0119] 9. Waxman, S., Ed., Differentiation Therapy, Challenges inMolecular Medicine, Vol. 10, Ares-Serono Symposia Publications, Rome,Italy, 1995.

[0120] 10. Chellappan, S. P., Giordano, A., and Fisher, P. B., The roleof cyclin dependent kinases and their inhibitors in cellulardifferentiation and development. Current Topics in Microbiology andImmunology, Springer-Verlag, NY, in press, 1996.

[0121] 11. Jiang, H., Lin, J., and Fisher, P. B., A molecular definitionof terminal differentiation in human melanoma cells. Mol. Cell.Different., 2 (3): 221-239, 1994.

[0122] 12. Jiang, H., Lin, J., Su, Z.-z., and Fisher, P. B., Themelanoma differentiation associated gene-6 (mda-6), which encodes thecyclin-dependent kinase inhibitor p21, may function as a negativeregulator of human melanoma growth and progression. Mol. Cell.Different., 4 (1):67-89, 1996.

[0123] 13. Fisher, P. B., Prignoli, D. R., Hermo, H., Jr., Weinstein, I.B., and Pestka, S., Effects of combined treatment with interferon andmezerein on melanogenesis and growth in human melanoma cells. J.Interferon Res., 5: 11-22, 1985.

[0124] 14. Jiang, H., Su, Z.-z., Boyd, J., and Fisher, P. B., Geneexpression changes associated with reversible growth suppression and theinduction of terminal differentiation in human melanoma cells. Mol.Cell. Different., 1 (1): 41-66, 1993.

[0125] 15. Jiang, H., Lin, J., Young, S.-m., Goldstein, N. I., Waxman,S., Davila, V., Chellappan, S. P., and Fisher, P. B., Cell cycle geneexpression and E2F transcription factor complexes in human melanomacells induced to terminally differentiate. Oncogene, 11: 1179-1189,1995.

[0126] 16. Jiang, H., and Fisher, P. B., Use of a sensitive andefficient subtraction hybridization protocol for the identification ofgenes differentially regulated during the induction of differentiationin human melanoma cells. Mol. Cell. Different., 1 (3): 285-299, 1993.

[0127] 17. Jiang, H., Lin, J., Su, Z.-z., Herlyn, M., Kerbel, R. S.,Weissman, B. E., Welch, D. R., and Fisher, P. B., The melanomadifferentiation-associated gene mda-6, which encodes thecyclin-dependent kinase inhibitor p21, is differentially expressedduring growth, differentiation and progression in human melanoma cells.Oncogene, 10: 1855-1864, 1995.

[0128] 18. Jiang, H., Lin, J. J., Su, Z.-z., Goldstein, N. I., andFisher, P. B., Subtraction hybridization identifies a novel melanomadifferentiation associated gene, mda-7, modulated during human melanomadifferentiation, growth and progression. Oncogene, 11: 2477-2486, 1995.

[0129] 19. Jiang, H., Lin, J. J., Tao, J., and Fisher, P. B.,Suppression of human ribosomal protein L23A expression during cellgrowth inhibition by interferon-β. Oncogene, 14, in press, 1997.

[0130] 20. Jiang, H., Lin, J., Su, Z.-z., Collart, F. R., Huberman, E.,and Fisher, P. B., Induction of differentiation in human promyelocyticHL-60 leukemia cells activates p21, WAF1/CIP1, expression in the absenceof p53. Oncogene, 9:3397-3406, 1994.

[0131] 21. Giovanella, B. C., Stehlin, J. S., Santamaria, C., Yim, S.O., Morgan, A. C., Williams, L. J., Leibovitz, A., Fialkow, P. Y., andMumford, D. M., Human neoplastic and normal cells in tissue culture. I.Cell lines derived from malignant melanomas and normal melanocytes. J.Natl. Cancer Inst., 56: 1131-1142, 1976.

[0132] 22. Huberman, E., Heckman, C., and Langenbach, R., Stimulation ofdifferentiated functions in human melanoma cells by tumor-promotingagents and dimethyl sulfoxide. Cancer Res., 39: 2618-2624, 1979.

[0133] 23. Melber, K., Zhu, G., and Diamond, L., SV40-transformed humanmelanocyte sensitivity to growth inhibition by the phorbol ester12-0-tetradecanoylphorbol-13-acetate. Cancer Res., 49: 3650-3655, 1989.

[0134] 24. Herlyn, M., Human melanoma: development and progression.Cancer Metastasis Rev., 9: 101-112, 1990.

[0135] 25. Frohman, M. A., Dush, M. K, and Martin, G. R., Rapidproduction of full-length cDNAs from rare transcripts: amplificationusing a single gene-specific oligonucleotide primer. Proc. Natl. Acad.Sci. U.S.A., 85: 8998-9002, 1988.

[0136] 26. Loh, E. Y., Elliot, J. F., Cwirla, S. A., Lanier, L. L., andDavis, M. M., Polymerase chain reaction with single-sided specificity:analysis of T cell receptor delta chain. Science, 243: 217-220, 1989.

[0137] 27. Ohara, O., Dorit, R. L., and Gilbert, W., Direct genomicsequencing of bacterial DNA: the pyruvate kinase 1 gene of Escherichiacoli. Proc. Natl. Acad. Sci. U.S.A., 86: 6883-6887, 1989.

[0138] 28. Sanger, F., Nicklen, S., and Coulson, A. R., DNA sequencingwith chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A., 74:5463-5467, 1977.

[0139] 29. Su, Z.-z., Leon, J. A., Jiang, H., Austin, V. A., Zimmer, S.G., and Fisher, P. B., Wild-type adenovirus type 5 transforming genesfunction as trans-dominant suppressors of oncogenesis in mutantadenovirus type 5 transformed rat embryo fibroblast cells. Cancer Res.,53: 1929-1938, 1993.

[0140] 30. Reddy, P. G., Graham, G. M., Datta, S., Guarini, L., Moulton,T. A., Jiang, H., Gottesman, M. M., Ferrone, S., and Fisher, P. B.,Effect of recombinant fibroblast interferon and recombinant immuneinterferon on growth and the antigenic phenotype of multidrug-resistanthuman glioblastoma multiforme cell. J. Natl. Cancer Inst., 83:1307-1315, 1991.

[0141] 31. Su, Z.-z., Grunberger, D., and Fisher, P. B., Suppression ofadenovirus type 5 E1A-mediated transformation and expression of thetransformed phenotype by caffeic acid phenethyl ester (CAPE). Mol.Carcinog., 4: 231-242, 1991.

[0142] 32. Jiang, H., Su, Z.-z., Datta, S., Guarini, L., Waxman, S., andFisher, P. B., Fludarabine phosphate selectively inhibits growth andmodifies the antigenic phenotype of human glioblastoma multiforme cellsexpressing a multidrug resistance phenotype. Intl. J. Oncol., 1:227-239, 1992.

[0143] 33. Fort, P., Marty, L., Piechaczyk, M., Sabrouty, S. E., Dani,C., Jeanteur, P., and Blanchard, J. M., Various rat adult tissuesexpress only one major mRNA species from theglyceraldehyde-3-phosphate-dehydrogenase multigenic family. NucleicAcids Res., 13: 1431-1442, 1985.

[0144] 34. Jiang, H., Waxman, S., and Fisher, P. B., Regulation ofc-fos, c-jun, and jun-B gene expression in human melanoma cells inducedto terminally differentiate. Mol. Cell. Different., 1 (2): 197-214,1993.

[0145] 35. Mark, D. B., Lu, S. D., Creasey, A., Yamamoto, R., and Lin,L., Site-specific mutagenesis of the human fibroblast interferon gene.Proc. Natl. Acad. Sci. U.S.A., 81: 5662-5666, 1984.

[0146] 36. Rehberg, G., Kelder, B., Hoal, E. G., and Pestka, S.,Specific molecular activities of recombinant and hybrid leukocyteinterferons. J. Biol. Chem., 257: 11497-11502, 1982.

[0147] 37. Duclos, F., Boschert, U., Sirugo, G., Mandel, J.-L., Hen, R.,and Koenig, M., Gene in the region of the Friedreich ataxia locusencodes a putative transmembrane protein expressed in the nervoussystem. Proc. Natl. Acad. Sci. U.S.A., 90: 109-113, 1993.

[0148] Second Series of Experiments

[0149] Subtraction hybridization using a cDNA library prepared fromtemporally spaced mRNAs from human melanoma cells treated withrecombinant human fibroblast interferon (IFN-β) plus mezerein (MEZ) thatinduces terminal differentiation (tester cDNA library) and a temporallyspaced cDNA library prepared from actively proliferating melanoma cells(driver cDNA library) produced a Temporally Spaced Subtracted (TSS) cDNAlibrary. This approach resulted in the identification of melanomadifferentiation associated (mda) genes displaying both enhanced andsuppressed expression during growth inhibition and differentiation. Inthe present report, we describe a novel cDNA mda-9 that consists of2,084 nucleotides, and encodes a protein of 298 amino acids with apredicted M_(r) of ˜33 kDa. Treatment of human SV40-immortalized normalmelanocytes, early radial growth phase primary melanoma and metastaticmelanoma cells with immune interferon, IFN-γ, induces growth suppressionand enhances mda-9 expression without inducing terminal differentiation.These results establish that induction of terminal differentiation inhuman melanoma cells, using the combination of a type I interferon(IFN-β)+MEZ, can elicit signaling pathways and gene expression changesalso regulated by type II immune interferon.

[0150] Treatment of metastatic human melanoma cells with the combinationof IFN-β and the antileukemic compound MEZ results in an irreversibleloss of proliferative ability and terminal cell differentiation (Fisheret al., 1985; Jiang et al., 1993). To define the gene expression changesthat mediate these phenomena we have used an efficient subtractionhybridization approach (Jiang and Fisher, 1993; Jiang et al., 1995a,1995b). Since the precise spatial kinectics of the specific geneexpression changes involved in inducing terminal differentiation inchemically treated H0-1 human melanoma cells are not known, our cloningstrategy involved the use of pooled mRNAs collected at various timesafter treatment with IFN-β+MEZ, i.e., 2, 4, 8, 12 and 24 hr, toconstruct a tester cDNA library. A second cDNA library was generatedfrom mRNAs isolated over the same time periods from actively growingH0-1 human melanoma cells and this temporally spaced driver cDNA librarywas subtracted from the temporally spaced tester cDNA library, therebyproducing a differentiation-inducer treated TSS human melanoma cDNAlibrary (Jiang and Fisher, 1993; Lin et al., 1996).

[0151] Seventy random cDNA clones were isolated from thedifferentiation-inducer treated TSS human melanoma cDNA library(representing ˜2.5% of the total subtracted cDNA library) resulting in23 clones displaying elevated expression after exposure to the inducingagent(s), IFN-β, MEZ and/or IFN-β+MEZ (Jiang and Fisher, 1993). Thisapproach resulted in the cloning of 9 originally novel mda genes, notpreviously reported in DNA data bases. Subsequent studies indicate thatthe unique mda-6 gene which is upregulated during growth arrest andterminal differentiation in human melanoma cells is identical to thecyclin dependent kinase inhibitor p21, also identified as Waf1, Cip1 andSdi1 (Jiang et al., 1994, 1995b, 1996a). A novel gene mda-7 that is alsoelevated in terminally differentiated human melanoma cells is aubiquitous inhibitor of cancer growth (Jiang et al., 1995b, 1996b). Themda-20 gene is the human ribosomal protein L23a gene that isdown-regulated following treatment with IFN-β (Jiang et al., 1997). Thenovel mda-9 gene displays decreased expression as a function ofinduction of irreversible growth suppression and terminaldifferentiation in several human melanoma cell lines (Lin et al., 1996).

[0152] In the present study we demonstrate that mda-9 expression iselevated following IFN-γ treatment in human melanocyte and melanoma celllines, even though growth is suppressed. This observation isunanticipated and suggests that type II interferon responsive genes maybe up-regulated as a function of induction of terminal differentiationby human IFN-β (a product of the type I interferon gene) in combinationwith MEZ. Furthermore, the direction of a specific gene expressionchange in growth suppressed human melanoma is dependent on the inducingagent and is subject to either increased or decreased expression. Inthis context, terminal differentiation clearly involves complex pathwayswith overlapping convergent and divergent gene expression changes thatcan also be elicited after exposure to specific cytokines, such asIFN-γ.

[0153] Experimental Details

[0154] Materials and Methods

[0155] Cell lines and culture conditions. H0-1 is a melanotic melanomacell line produced from a metastatic inguinal node lesion from a49-year-old female and was used between passages 170 and 200 (Giovanellaet al., 1976; Huberman et al., 1979; Fisher et al., 1985). C8161 is ahighly metastatic amelanotic human melanoma cell line derived from anabdominal wall metastasis (Welch et al., 1991). FM516-SV is a normalhuman melanocyte culture immortalized by the SV40 T-antigen gene (Melberet al., 1989). The WM35 cell line was isolated from a patient with aradial growth phase primary melanoma (Herlyn, 1990). The WM278 cell lineis from a patient with an early vertical growth phase primary melanoma(Herlyn, 1990). Cultures were grown at 37° C. in a 95% air 5%C0₂-humidified incubator in Dulbecco's modified Eagle's medium (DMEM)supplemented with 5 or 10% fetal bovine serum. Cultures were maintainedin the logarithmic phase of growth by subculturing (1:5 or 1:10) priorto confluence approximately every 4 to 7 D.

[0156] Subtraction hybridization, RACE and sequence analysis.Identification and cloning of mda-9 was accomplished as describedpreviously (Jiang and Fisher, 1993). Briefly, a cDNA library wasprepared from RNA isolated from actively growing H0-1 cells (driver) andRNAs isolated from H0-1 cells treated with IFN-β+MEZ (2000 U/ml+10ng/ml) for 2, 4, 8, 12, and 24 hr (temporally spaced tester).Subtraction hybridization was then performed between double-strandedtester DNA and single-stranded driver DNA prepared by mass excision ofthe libraries. The TSS cDNAs were efficiently cloned into the λ Uni-ZAPphage vector and used to screen Northern blots containing total RNAisolated from control H0-1 cells and H0-1 cultures treated for 24 hrwith IFN-β (2000 U/ml), MEZ (10 ng/ml) or IFN-β+MEZ (2000 U/ml+10ng/ml). This strategy resulted in the identification of a partial mda-9cDNA (Jiang et al., 1994). A full-length mda-9 cDNA was isolatedfollowing screening of a differentiation inducer-treated H0-1 cDNAlibrary (Jiang and Fisher, 1993) and using the procedure of rapidamplification of cDNA ends (RACE) as described previously (Jiang et al.,1995b). Sequence analysis was determined as described (Sanger et al.,1977).

[0157] RNA isolation and northern blotting. Total cellular RNA wasisolated by the guanidinium/phenol procedure, and Northern blotting wasperformed as described (Reddy et al., 1991; Su et al., 1991). Tenmicrograms of RNA were denatured with glyoxal/dimethyl sulfoxide (DMSO),electrophoresed in 1.0% agarose gels, transferred to nylon membranes,and hybridized with a ³²P-labeled mda-9 probe. After stripping themembranes were hybridized to a ³²P-labeled rat GAPDH probe (Fort et al.,1985) as described previously (Reddy et al., 1991; Su et al., 1991).Following hybridization, the filters were washed and exposed forautoradiography. RNA blots were quantitated by densitometric analysisusing a Molecular Dynamics densitometer (Sunnyvale, Calif.) (Jiang etal., 1993).

[0158] Recombinant human gamma interferon. Recombinant human IFN-γ waskindly provided by Dr. Sidney Pestka (UMDNJ-Robert Wood Johnson MedicalSchool, NJ). The interferon titer was determined using a cytopathiceffect inhibition assay with VSV on the human WISH cell line (Rehberg etal., 1982).

[0159] Experimental Results

[0160] Subtraction hybridization identifies mda-9 as a component of theterminal differentiation pathway in human melanoma cells.

[0161] Subtraction hybridization represents an effective experimentalapproach for identifying and cloning genes displaying differentialexpression. This strategy has been applied to human melanoma cellsinduced to terminally differentiate by treatment with IFN-β+MEZresulting in the cloning of mda genes (Jiang and Fisher, 1993). Wepresently describe properties of mda-9, a novel cDNA of 2,084nucleotides (Lin et al., 1996) with sequence homology to a recentlyreported human scaffold protein Pbp1 gene (U83463; deposited Jan. 1,1997) (FIG. 7A) (Accession # AF006636). In vitro translation confirmsthat mda-9 encodes a putative protein with a predicted M_(r) of ˜33 kDa(data not shown). Tissue distribution analysis indicates that mda-9 iswidely expressed in diverse tissues, with slightly elevated expressionin brain (putamen) and spleen (adult and fetal) (Lin et al., 1996).Southern blot analysis documents that mda-9 is a well-conserved genewith a homologous sequence present in yeast (Lin et al., 1996).

[0162] With the aid of the GCG computer package (Genetics ComputerGroup, Madison, Wis.), the mda-9 sequence was analyzed. An open readingframe (ORF) is designated to a region from nucleotide (nt) 76 to 972which starts at a methionine codon located in the context relative tothe Kozak consensus sequence (Kozak, 1987). The ORF encodes a 298 aminoacid (aa) protein (FIG. 7) with a predicted mass of 32.48 kDa asconfirmed by an in vitro translation assay (data not shown). Thepredicted mda-9 protein contains two possible protein kinase C (PKC)phosphorylation sites at aa 171 and 189, five possible casein kinase IIphosphorylation sites at aa 6, 60, 97, 189, 289, and 294, one possibletyrosine phosphorylation site at aa 48, and seven possible myristylationsites at aa 58, 80, 98, 102, 151, 248, and 262. A transmembrane segmentfrom aa 257 to 276 can be anticipated based on the strong tendency ofthe amino acid sequence to form transmembrane helices. No homology toother motifs were found. Recently, a cDNA sequence was deposited inGenbank as a scaffold protein (Pbp1) (U83463), that is identical tomda-9 except that it is shorter than the mda-9 cDNA by 87 bp. Noreference to the putative function of the Pbp1 gene is provided.

[0163] The mda-9 ORF is flanked by 5′ untranslated region of 75 bp and3′ untranslated region of 1096 bp, respectively. There is no additionalATG codon in the 5′ untranslated region. We were unable to obtain cDNAswith longer 5′-leader sequences by the RACE technique, suggesting thatthe 5′-end of the mda-9 sequence is the transcription initiation site.The 3′ untranslated region contains one consensus element (ATTTA, nt1390) involved in mRNA instability (Shaw and Kamen, 1986). No match tothe consensus poly (A) signal sequence (AATAAA) is present, suggestingthat a variant of the polyadenylation signal, AATTAA at nt 2051, servesthis function (Wickens and Stephenson, 1984).

[0164] Effect of IFN-γ on Melanocyte and Melanoma Cell Growth andExpression of mda-9

[0165] IFN-γ inhibits the growth of SV40-transformed normal humanmelanocyte and human melanoma cells (Table 2). With specific cell types,even 1 U/ml of IFN-γ induces growth suppression. The proliferationinhibitory effects of IFN-γ are most pronounced in the melanocyte(FM516-SV) and the radial growth phase primary melanoma (WM35) celllines (Table 2). In contrast to its effect on cell growth, IFN-γvariably stimulates mda-9 expression in the different cell lines.Increases in RNA levels range from ˜1.7-fold to ˜23.7-fold and varybetween cell types and in different experiments (FIG. 8 and data notshown). No direct relationship is apparent between the degree of IFN-γinduced growth inhibition and the relative level of increase in mda-9mRNA expression. Enhanced mda-9 expression is apparent within 2 hr ofexposure of H0-1 cells to 100 U/ml of recombinant human IFN-γ (FIG. 9A).The maximum increase in mda-9 RNA in H0-1 cells following IFN-γtreatment occurs between 8 and 24 hr post-treatment. A similar patternof increase is apparent in FMS16-SV cells treated with 100 U/ml of IFN-γ(data not shown). IFN-γ dose-response studies in humanmelanocyte/melanoma cells indicate that mda-9 regulation is extremelysensitive to this cytokine (FIG. (9B). Even with doses as low as 0.1U/ml, mda-9 expression is elevated in H0-1 (FIG. 9B) and FM516-SV (datanot shown) cells. Maximum enhancing effects are evident when H0-1 orFM516-SV cells are treated with 10 U/ml of IFN-γ. TABLE 2 Effect ofrecombinant Immune Interferon (IFN-γ) on the growth of normal humanmelanocyte and melanoma cell lines Experimental Cell Lines AnalyzedConditions^(a) FM516-SV WM35 WM278 H0-1 C8161 Control 20.7 ± 0.6 86.5 ±2.1 22.0 ± 1.0 20.3 ± 1.7 34.8 ± 0.7 1 16.8 ± 0.5 (19) 84.8 ± 6.7 (2)14.9 ± 0.8 (33) 15.8 ± 0.5 (22) 24.4 ± 0.8 (30) 10  9.8 ± 0.6 (52) 62.7± 4.3 (28) 12.7 ± 1.0 (42) 15.4 ± 0.3 (24) 25.1 ± 0.8 (28) 100  5.1 ±0.3 (75) 28.2 ± 1.7 (67) 12.5 ± 0.6 (44) 11.5 ± 1.3 (44) 22.2 ± 1.4 (36)# immortalized by SV40; WM35 is a cell line produced from a patient withan early radial growth phase primary human melanoma; WM278, H0-1 andC8161, are cell lines established from metastatic human melanoma.

[0166] Experimental Discussion

[0167] Subtraction hybridization with temporally spaced mRNA samplespermitted cloning of a novel gene, mda-9, that displays biphasicregulation during induction of irreversible growth suppression andterminal differentiation in human melanoma cells (Lin et al., 1996).Treatment of H0-1 cells with IFN-β+MEZ results in maximum increases inmda-9 RNA 8 to 12 hr post treatment and a reduction in mda-9 expressionat 24 hr. The present study demonstrates that mda-9 is variablyupregulated by recombinant human IFN-γ in SV40-immortalized humanmelanocyte and human melanoma cells even though growth is suppressed.Maximum mda-9 levels are observed between 8 and 24 hr treatment withIFN-γ. These results document that the expression of mda-9 is subject tocomplex regulation in human melanocyte/melanoma lineage cells and thedirection of the response, i.e., increase or decrease, is dependent onthe inducing agent and the cellular program modified.

[0168] Recent studies are providing important insights into thesignaling pathways involved in IFN-γ regulation of gene expression(Darnell et al., 1994; Bach et al., 1997; Boehm et al., 1997). Thecurrent model for IFN-γ induction of gene expression involves binding ofIFN-γ to high affinity cell surface receptors, transphosphorylation andactivation of JAK1 and JAK2 kinases and phosphorylation of STAT (signaltransducers and activators of transcription) proteins. Thephosphorylated STAT proteins migrate into the nucleus, binding tospecific DNA elements (gamma-interferon activation site; GAS, containing9 nucleotides with a consensus sequence of TTNCNNNAA) and directtranscriptional activation of IFN-γ-inducible genes. Further studies arerequired to determine if enhanced mda-9 expression occurs via thispathway following treatment with IFN-γ and/or during the process ofinduction of terminal differentiation by treatment with IFN-β+MEZ.

[0169] The molecular determinants of growth control and terminaldifferentiation in human melanoma cells are beginning to be defined(Jiang et al., 1994). Mda-9 may represent an important element in theseprocesses. Moreover, elucidation of the role of mda-9 in growth control,terminal differentiation and response to specific cytokines (such asIFN-γ) will prove valuable in mechanistically understanding theseimportant physiological processes.

REFERENCES FOR SECOND SERIES OF EXPERIMENTS

[0170] Bach, E. A., Aguet, M. and Schreiber, R. D. (1997) The IFNγreceptor: a paradigm for cytokine receptor signaling. Annu. Rev.Immunol. 15, 563-591.

[0171] Boehm, U., Klamp, T., Groot, M. and Howard, J. C. (1997) Cellularresponses to interferon-γ. Annu. Rev. Immunol. 15, 749-795.

[0172] Darnell, J. E., Kerr, I. M. and Stark, G. R. (1994) Jak-STATpathways and transcriptional activation in response to IFNs and otherextracellular signaling proteins. Science 264, 1415-1421.

[0173] Fisher, P. B., Prignoli, D. R., Hermo, H., Jr., Weinstein, I. B.and Pestka, S. (1985) Effects of combined treatment with interferon andmezerein on melanogenesis and growth in human melanoma cells. J.Interferon Res. 5, 11-22.

[0174] Fort, P., Marty, L., Piechaczyk, M., Sabrouty, S. E., Dani, C.,Jeanteur, P. and Blanchard, J. M. (1985) Various rat adult tissuesexpress only one major mRNA species from theglyceraldehyde-3-phosphate-dehydrogenase multigenic family. NucleicAcids Res. 13, 1431-1442.

[0175] Giovanella, B. C., Stehlin, J. S., Santamaria, C., Yim, S. O.,Morgan, A. C., Williams, L. J., Leibovitz, A., Fialkow, P. Y., andMumford, D. M. (1976) Human neoplastic and normal cells in tissueculture. I. Cell lines derived from malignant melanomas and normalmelanocytes. J. Natl. Cancer Inst. 56, 1131-1142.

[0176] Herlyn, M. (1990) Human melanoma development and progression.Cancer Metastasis Rev. 9, 101-112.

[0177] Huberman, E., Heckman, C., and Langenbach, R. (1979) Stimulationof differentiated functions in human melanoma cells by tumor-promotingagents and dimethyl sulfoxide. Cancer Res. 39, 2618-2624.

[0178] Jiang, H. and Fisher, P. B. (1993) Use of a sensitive andefficient subtraction hybridization protocol for the identification ofgenes differentially regulated during the induction of differentiationin human melanoma cells. Mol. Cell. Different. 1 (3), 285-299.

[0179] Jiang, H., Lin, J., and Fisher, P. B. (1994) A moleculardefinition of terminal differentiation in human melanoma cells. Mol.Cell. Different. 2 (3) 221-239.

[0180] Jiang, H., Lin, J., Su, Z.-z. and Fisher, P. B. (1996a) Themelanoma differentiation associated gene-6 (mda-6), which encodes thecyclin-dependent kinase inhibitor p21, may function as a negativeregulator of human melanoma growth and progression. Mol. Cell.Different. 4 (1), 67-89.

[0181] Jiang, H., Lin, J. J., Su, Z.-z., Goldstein, N. I. and Fisher, P.B. (1995a) Subtraction hybridization identifies a novel melanomadifferentiation associated gene, mda-7, modulated during human melanomadifferentiation, growth and progression. Oncogene 11, 2477-2486.

[0182] Jiang, H., Lin, J., Su, Z.-z., Herlyn, M., Kerbel, R. S.,Weissman, B. E., Welch, D. R. and Fisher, P. B. (1995b) The melanomadifferentiation-associated gene mda-6, which encodes thecyclin-dependent kinase inhibitor p21, is differentially expressedduring growth, differentiation and progression in human melanoma cells.Oncogene 10, 1855-1864.

[0183] Jiang, H., Lin, J. J., Tao and Fisher, P. B. (1997) Suppressionof human ribosomal protein L23A expression during cell growth inhibitionby interferon-β. Oncogene 14, 473-480.

[0184] Jiang, H., Su, Z.-z., Boyd, J. and Fisher, P. B. (1993) Geneexpression changes associated with reversible growth suppression and theinduction of terminal differentiation in human melanoma cells. Mol.Cell. Different. 1 (1): 41-66.

[0185] Jiang, H., Su, Z.-z., Lin, J. J., Goldstein, N. I., Young, C. S.H. and Fisher, P. B. (1996b) The melanoma differentiation associatedgene mda-7 suppresses cancer cell growth. Proc. Natl. Acad. Sci. USA 93:9160-9165.

[0186] Jiang, H., Waxman, S. and Fisher, P. B. (1993) Regulation ofc-fos, c-jun, and Jun-B gene expression in human melanoma cells inducedto terminally differentiate. Mol. Cell. Different. 1 (2): 197-214.

[0187] Kozak, M. (1987) An analysis of 5′-noncoding sequences from 699vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.

[0188] Lin, J. J., Jiang, H. and Fisher, P. B. (1996) Characterizationof a novel melanoma differentiation associated gene, mda-9, that isdown-regulated during terminal cell differentiation. Mol. Cell.Different. 4 (4): 317-333.

[0189] Melber, K., Zhu, G. and Diamond, L. (1989) SV40-Transformed humanmelanocyte sensitivity to growth inhibition by the phorbol ester12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 49, 3650-3655.

[0190] Reddy, P. G., Graham, G. M., Datta, S., Guarini, L., Moulton, T.A., Jiang, H., Gottesman, M. M., Ferrone, S. and Fisher, P. B. (1991)Effect of recombinant fibroblast interferon and recombinant immuneinterferon on growth and the antigenic phenotype of multidrug-resistanthuman glioblastoma multiforme cells. J. Natl. Cancer Inst. 83,1307-1315.

[0191] Rehberg, G., Kelder, B., Hoal, E. G. and Pestka, S. (1982)Specific molecular activities of recombinant and hybrid leukocyteinterferons. J. Biol. Chem. 257, 11497-11502.

[0192] Sanger, F., Nicklen, S. and Coulson, A. R. (1977) DNA sequencingwith chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467.

[0193] Su, Z.-Z., Grunberger, D. and Fisher, P. B. (1991) Suppression ofadenovirus type 5 E1A-mediated transformation and expression of thetransformed phenotype by caffeic acid phenethyl ester (CAPE). Mol.Carcinog. 4, 231-242.

[0194] Shaw, G. and Kamen, R. (1986). A consensus AU sequence form the3′ untranslated region of GM-CSF mRNA mediates selective mRNAdegradation. Cell, 46, 659-667.

[0195] Welch, D. R., Bisi, J. E. Miller, B. E., Conaway, D., Seftor, E.A., Yokem, K. H., Gilmore, L. B., Seftor, R. E., Nakajima, M. andHendrix, M. J. (1991) Characterization of a highly invasive andspontaneously metastatic human malignant melanoma cell line. Intl. J.Cancer, 47, 227-237.

[0196] Wickens, M. and Stephenson, P. (1984). Role of the conservedAAUAAA sequence: four AAUAAA point mutants prevent messenger RNA 3′ endformation. Nature, 226, 1045-1051.

[0197] Third Series of Experiments

[0198] Previous experiments demonstrate that mda-9 expression occurs in50 normal human tissues and various human cancer cell lines (1,2).Induction of terminal differentiation with an irreversible loss inproliferative ability in human melanoma cells by treatment withrecombinant human fibroblast interferon (IFN-β) plus the antileukemiccompound mezerein (MEZ) (3,4) results in a reduction in mda-9 expression(1) In contrast, treatment of the same cell types with variousinterferons, including IFN-β, recombinant human leukocyte interferon(IFN-α) and recombinant human immune interferon (IFN-γ), results ingrowth suppression and an elevation in mda-9 expression (1,2). Thegrowth suppression effect is greatest with IFN-β, whereas theenhancement in mda-9 gene expression is greatest with IFN-γ. Theseresults demonstrate that mda-9 expression can be modified by a number ofdiverse signals, including induction of differentiation and treatmentwith recombinant cytokines, that occur in a growth-independent manner.

[0199] To directly define a functional role for mda-9 in cellularphysiology, pREP4 expression vectors were constructed that express mda-9in either a sense orientation (pREP-mda-9 S) or an antisense orientation(pREP-mda-9 AS). Constructs were prepared as described previously inJiang et al. (5-7). Experiments were then performed to determine theeffect of altering mda-9 expression on the growth of HBL-100, animmortalized normal human breast epithelial cell line (containing SV40sequences) and MCF-7, a human breast carcinoma cell line. The results ofa representative study determining the effect of pREP4 (expressionvector construct lacking the mda-9 gene), pREP-mda-9 S and pREP-mda-9 ASare shown in Table 3. In both cell types, forced expression of mda-9 ineither a sense or antisense orientation inhibited colony formation.Inhibition was greatest in the two cell types when treated with ASmda-9. The effect on growth was greater in HEL-100 than in MCF-7 cells.

[0200] The studies briefly described above demonstrate that mda-9directly contributes to cell growth. Interestingly, since bothoverexpression (mda-9 S) and inhibition in expression (mda-9 AS)modifies growth, it appears that mda-9 may function as a barometer formaintaining cellular homeostasis. If this hypothesis is correctstrategies designed to modify mda-9 expression during treatment withvarious compounds may permit the use of this gene for therapeuticapplications. For example, combining AS mda-9 expression (using atargeting vector such as an adenovirus, adeno-associated virus,retrovirus, vaccinia virus, Epstein Barr virus, etc.)(7,8) with agentscapable of partially inducing differentiation, such as IFN-β or MEZ, butnot terminal differentiation may permit the use of a single agent fordifferentiation therapy of cancer. Moreover, combining S mda-9expression (using a targeting vector such as an adenovirus,adeno-associated virus, retrovirus, vaccinia virus, Epstein Barr virus,etc.) (7,8) in combination with specific cytokines, such as IFN-α,IFN-β, IFN-γ or TNF-α, DNA damaging chemotherapeutic agents (such asdactinomycin, cis-platinum, taxol and its analogs, etc.) or physicaltherapeutic agents (such as gamma irradiation) may result in enhancedgrowth suppression in tumor cells. These effects can be furtheraugmented and targeted to specific cell types by using gene expressionenhancers and tissue specific promoters. Experiments to confirm thesepossibilities are presently in progress. TABLE 3 Effect ofoverexpression and an inhibition in expression of mda-9 on colonyformation in monolayer culture of HBL-100 and MCF-7 cells. PlasmidColony Cell Type¹ Transfect² Numbers³ % of Control⁴ HBL-100 pREP4 913.75± 178.4 100 pREP-mda-9 S 258.5 ± 44.4  28.3 PREP-mda-9 AS 129.0 ± 30.3 14.1 MCF-7 pREP4 232.5 ± 12.5  100 pREP-mda-9 S 232.5 ± 16.1  80.7PREP-mda-9 AS 134.75 ± 10.3  58.0

REFERENCES FOR THIRD SERIES OF EXPERIMENTS

[0201] 1. Lin, J. J., Jiang, H., and Fisher, P. B. (1996)Characterization of a novel melanoma differentiation associated gene,mda-9, that is down-regulated during terminal cell differentiation. Mol.Cell. Different., 4(4):317-333.

[0202] 2. Lin, J. J., Jiang, H., and Fisher (197) Melanomadifferentiation associated gene-9, mda-9, is a human gamma interferonresponsive gene. Gene, in press.

[0203] 3. Fisher, P. B., Prignoli, D. R., Hermo, H., Jr., Weinstein, I.B. and Pestka, S. (1985) Effects of combined treatment with interferonand mezerein on melanogenesis and growth in human melanoma cells. J.Interferon Res. 5:11-22.

[0204] 4. Jiang, H., Su, Z.-z., Boyd, J., and Fisher, P. B. (1993) Geneexpression changes associated with reversible growth suppression and theinduction of terminal differentiation in human melanoma cells. Mol.Cell. Different., 1(1):41-66.

[0205] 5. Jiang, H., Lin, J., Su, Z-z., Herlyn, M., Kerbel, R. S.,Weissman, B. E., Welch., D. R., and Fisher, P. B. (1995) The melanomadifferentiation-associated gene mda-7, which encodes thecyclin-dependent kinase inhibitor p21, is differentially expressedduring growth, differentiation and progression in human melanoma cell.Oncogene, 10:1855-1864.

[0206] 6. Jiang, H., Lin., J. J., Su, Z.-z., Goldstein, N. I., andFisher, P. B. (1995) Substraction hybridization identifies a novelmelanoma differentiation associated gene, mda-7, modulated during humanmelanoma differentiation, growth and progression. Oncogene,11:2477-2486.

[0207] 7, Jiang, H. Su, Z.-z., Lin, J. J., Goldstein, N. I., Young C. S.H., and Fisher, P. B. (1996). The melanoma differentiation associatedgene mda-7 suppresses cancer cell growth. Proc. Natl. Acad. Sci. USA,93:9160-9165.

[0208] 8. Su, Z.-z., Madireddi, M. T., Lin, J. J., Young, C. S. H.,Kitada, S., Reed, J. C., Goldstein, N. I. and Fisher, P. B. (1997) Thecancer growth suppressor gene mda-7 selectively induces apoptosis inhuman cancer cells and inhibits tumor growth in nude mice. Inpreparation.

[0209]

1 2 1 2084 DNA Homo sapiens 1 cctcagaagt ccgtgccagt gaccggaggcggcggcggcg agcggttcct tgtgggctag 60 aagaatcctg caaaaatgtc tctctatccatctctcgagg acttgaaggt aaacaaatta 120 attcaggctc aaactgcttt ttctgcaaaccctgccaatc cagcaatttt gtcagaagct 180 tctgctccta tccctcacga tggaaatctctatcccagac tgtatccaga gctctctcaa 240 tacatggggc tgagtttaaa tgaagaagaaatacgtgcaa atgtggccgt ggtttctggt 300 gcaccacttc aggggcagtt ggtagcaagaccttccagta taaactatat ggtggctcct 360 gtaactggta atgatgttgg aattcgtagagcagaaatta agcaagggat tcgtgaagtc 420 attttgtgta aggatcaaga tggaaaaattggactcaggc ttaaatcaat agataatggt 480 atatttgttc agctagtcca ggctaattctccagcctcat tggttggtct gagatttggg 540 gaccaagtac ttcagatcaa tggtgaaaactgtgcaggat ggagctctga taaagcgcac 600 aaggtgctca aacaggcttt tggagagaagattaccatga ccattcgtga caggcccttt 660 gaacggacga ttaccatgca taaggatagcactggacatg ttggttttat ctttaaaaat 720 ggaaaaataa catccatagt gaaagatagctctgcagcca gaaatggtct tctcacggaa 780 cataacatct gtgaaatcaa tggacagaatgtcattggat tgaaggactc tcaaattgca 840 gacatactgt caacatctgg gactgtagttactattacaa tcatgcctgc ttttatcttt 900 gaacatatta ttaagcggat ggcaccaagcattatgaaaa gcctaatgga ccacaccatt 960 cctgaggttt aaaattcacg gcaccatggaaatgtagctg aacgtctcca gtttccttct 1020 ttggcaactt ctgtattatg cacgtgaagccttcccggag ccagcgagca tatgctgcat 1080 gaggaccttt ctatcttaca ttatggctgggaatcttact ctttcatctg ataccttgtt 1140 cagatttcaa aatagttgta gccttatcctggttttacag atgtgaaact ttcaagagat 1200 ttactgactt tcctagaata gtttctctactggaaacctg atgcttttat aagccattgt 1260 gattaggatg actgttacag gcttagctttgtgtgaaaac cagtcacctt tctcctaggt 1320 aatgagtagt gctgttcata ttactttagttctatagcat actgcatctt taacatgcta 1380 tcatagtaca tttagaatga ttgcctttgatttttttttt aaattctgtg tgtgtgtgtg 1440 taaaatgcca attaagaaca ctggtttcattccatgtaag cattaaacag tgtatgtagg 1500 tttcaagaga ttgtgatgat tcttaaattttaactacctt cacttaatat gcttgaactg 1560 tcgccttaac tatgttaagc atctagactaaaagccaaaa tataattatt gctgcctttc 1620 taaaaaccca aaatgtagtt ctctattaacctgaaatgta cactagccca gaacagttta 1680 atggtactta ctgagctata gcatagctgcttagttgttt ttgagagttt ttagtcaaca 1740 cataatggaa acttctttct tctaaaagttgccagtgcca cttttaagaa gtgaatcact 1800 atatgtgatg taaaagttat tacactaaacaggataaact tttgactccc cttttgttca 1860 tttgtggatt aagtggtata atacttaattttggcatttg actcttaaga ttatgtaacc 1920 tagctacttt gggatggtct tagaatatttttctgataac ttgttccttt tcctgactcc 1980 tccttgcaaa caaaatgata gttgacactttatcctgatt tttttcttct ttttggttta 2040 tgtctattct aattaaatat gtataaataaaaaaaaaaaa aaaa 2084 2 298 PRT Homo sapiens PHOSPHORYLATION(171)...(189) PKC phosphorylation 2 Met Ser Leu Tyr Pro Ser Leu Glu AspLeu Lys Val Asn Lys Leu Ile 1 5 10 15 Gln Ala Gln Thr Ala Phe Ser AlaAsn Pro Ala Asn Pro Ala Ile Leu 20 25 30 Ser Glu Ala Ser Ala Pro Ile ProHis Asp Gly Asn Leu Tyr Pro Arg 35 40 45 Leu Tyr Pro Glu Leu Ser Gln TyrMet Gly Leu Ser Leu Asn Glu Glu 50 55 60 Glu Ile Arg Ala Asn Val Ala ValVal Ser Gly Ala Pro Leu Gln Gly 65 70 75 80 Gln Leu Val Ala Arg Pro SerSer Ile Asn Tyr Met Val Ala Pro Val 85 90 95 Thr Gly Asn Asp Val Gly IleArg Arg Ala Glu Ile Lys Gln Gly Ile 100 105 110 Arg Glu Val Ile Leu CysLys Asp Gln Asp Gly Lys Ile Gly Leu Arg 115 120 125 Leu Lys Ser Ile AspAsn Gly Ile Phe Val Gln Leu Val Gln Ala Asn 130 135 140 Ser Pro Ala SerLeu Val Gly Leu Arg Phe Gly Asp Gln Val Leu Gln 145 150 155 160 Ile AsnGly Glu Asn Cys Ala Gly Trp Ser Ser Asp Lys Ala His Lys 165 170 175 ValLeu Lys Gln Ala Phe Gly Glu Lys Ile Thr Met Thr Ile Arg Asp 180 185 190Arg Pro Phe Glu Arg Thr Ile Thr Met His Lys Asp Ser Thr Gly His 195 200205 Val Gly Phe Ile Phe Lys Asn Gly Lys Ile Thr Ser Ile Val Lys Asp 210215 220 Ser Ser Ala Ala Arg Asn Gly Leu Leu Thr Glu His Asn Ile Cys Glu225 230 235 240 Ile Asn Gly Gln Asn Val Ile Gly Leu Lys Asp Ser Gln IleAla Asp 245 250 255 Ile Leu Ser Thr Ser Gly Thr Val Val Thr Ile Thr IleMet Pro Ala 260 265 270 Phe Ile Phe Glu His Ile Ile Lys Arg Met Ala ProSer Ile Met Lys 275 280 285 Ser Leu Met Asp His Thr Ile Pro Glu Val 290295

What is claimed is:
 1. A method for producing a temporally spacedsubtracted cDNA library comprising: a) isolating temporally spaced RNAsfrom cells; b) generating cDNA inserts from the RNAs isolated from step(a); c) producing a temporally spaced cDNA library having clonescontaining the cDNA inserts generated from step (b); d) producing doublestranded cDNA inserts from the temporally spaced cDNA library; e)denaturing the double stranded cDNA inserts; f) contacting the denatureddouble stranded cDNA inserts produced in step (e) with single-strandedDNAs from another cDNA library under conditions permitting hybridizationof the single-stranded DNAs and the double-stranded cDNA inserts; g)separating the hybridized cDNA inserts from the unhybridized inserts; h)generating a cDNA library of the unhybridized inserts, therebygenerating a temporally spaced subtracted cDNA library.
 2. A method ofclaim 1, wherein the cDNA library used to generate the single-strandedDNAs is from the same cell population as the cell population used togenerate the temporally spaced cDNA library.
 3. A method of claim 2,wherein the cDNA library allows propagation in single-stranded circleform.
 4. A method of claim 3, wherein the cDNA library is a λZAP cDNAlibrary.
 5. A method of claim 1, wherein the double stranded cDNAinserts in step (d) are produced by releasing double-stranded cDNAinserts from double-stranded cDNA clones of the temporally spaced cDNAlibrary with appropriate restriction enzymes.
 6. A method of claim 1,wherein the single-stranded cDNAs are labeled with biotin.
 7. A methodof claim 6, wherein the separating of step f) is performed by extractionwith streptavidin-phenol: chloroform.
 8. A method of claim 4, whereinthe cells are HO-1 human melanoma cells treated with IFN-β and MEZ.
 9. Amethod of claim 8, wherein the treatment with IFN-β and MEZ istemporally spaced.
 10. A method of claim 9, wherein the temporallyspaced treatment occurs at 2, 4, 8, 12, 24, and 48 hours.
 11. A methodof claim 10, wherein the single-stranded nucleic acid molecules are fromanother cDNA library of H0-1 melanoma cells.
 12. A method of claim 2,wherein the cells are terminally differentiated and the single-strandedcDNAs are from another cDNA library of undifferentiated cells.
 13. Amethod of claim 2, wherein the cells are undifferentiated and thesingle-stranded cDNAs are from another cDNA library of terminallydifferentiated cells.
 14. A method of claim 13, wherein the cells arecancerous cells.
 15. A method of claim 14, wherein the cancerous cellsare selected from a group consisting of melanoma cells, basal cellcarcinoma cells, squamous cell carcinoma cells, neuroblastoma cells,glioblastoma multiforme cells, myeloid leukemic cells, breast carcinomacells, colon carcinoma cells, endometrial carcinoma cells, lungcarcinoma cells, ovarian carcinoma cells, prostate carcinoma cells,cervical carcinoma cells, osteosarcoma cells and lymphoma cells.
 16. Amethod of claim 2, wherein the cells are induced to undergo reversiblegrowth arrest, DNA damage, or apoptosis and the single-stranded cDNAsare from another cDNA library of uninduced cells.
 17. A method of claim2, wherein the cells are uninduced cells and the single-stranded cDNAsare from cells induced to undergo reversible growth arrest, DNA damage,or apoptosis.
 18. A method of claim 2, wherein the cells are at onedevelopmental stage and the single-stranded cDNAs are from another cDNAlibrary of the cells at a different developmental stage.
 19. A method ofclaim 2, wherein the cells are cancerous and the single-stranded cDNAsare from another cDNA library from normal cells.
 20. A method of claim2, wherein the cells are from the skin, connective tissue, muscle,breast, brain, meninges, spinal cord, colon, endometrium, lung, prostateand ovary.
 21. A method of claim 1, further comprising introducing thesubtracted library into host cells.
 22. A method of claim 1, furthercomprising ligating the subtracted inserts into λ Uni-ZAP arms.
 23. Atemporally spaced subtracted library generated by the method of claim 1.24. A temporally spaced subtracted library generated by the method ofclaim
 11. 25. A method of identifying a melanoma differentiationassociated gene comprising: a) generating probes from clones of thetemporally spaced subtracted library of claim 24; and b) hybridizing theprobe with the total RNAs or mRNAs from H0-1 cells treated with IFN-βand MEZ and the total RNAs or mRNAs from untreated H0-1 cells,hybridization of the probe with the total RNAs or mRNAs from the treatedH0-1 cell but no, reduced, or enhanced hybridization with the total RNAsor mRNA from untreated cells indicating that the clone from which theprobe is generated carries a melanoma differentiation associated gene.26. A method of claim 25, wherein the mRNAs are probed with labeled cDNAclones generated from the temporally spaced subtracted library on a dotblot, hybridization of the probe with the mRNAs isolating a melanomadifferentiation associated gene.
 27. A melanoma differentiationassociated gene identified by the method of claim
 25. 28. A method ofidentifying temporally expressed genes from a single subtracted cDNAlibrary, comprising: a) cloning the cDNAs from the temporally spacedsubtracted cDNA library produced by the method of claim 1; b)hybridizing the clones obtained in step (a) with total RNAs isolatedfrom control and with RNAs from differentiation-inducer treated cells,hybridization of the probe RNAs from differentiation-inducer treatedcells, either enhanced or no or reduced hybridization with total RNAisolated from control cells indicating that the gene from which theprobe was isolated is temporally expressed, thereby identifyingtemporally expressed genes from a single subtracted cDNA library.
 29. Atemporally expressed gene identified by the method of claim
 28. 30. Amethod of claim 28, wherein the temporally expressed gene is cloned intoa λ ZAP phage vector.
 31. An isolated mda-9 gene.
 32. An isolatednucleic acid of claim 31, wherein the encoded mda-9 protein is a humanprotein.
 33. An isolated nucleic acid having the nucleic acid sequenceset forth in FIG. 7A.
 34. The isolated nucleic acid of claim 33, saidnucleic acid encoding a human protein, wherein the encoded human proteinis human mda-9.
 35. A human mda-9 protein having the amino acid sequenceset forth in FIG. 7B.
 36. A method for identifying a compound capable ofinducing terminal differentiation in cancer cells comprising: a)incubating an appropriate concentration of the cancer cells with anappropriate concentration of the compound; b) measuring the expressionof mda-9, the reduced expression of mda-9 gene indicating that thecompound is capable of inducing terminal differentiation in cancercells.
 37. A method of claim 36, wherein the cancer cells are selectedfrom a group consisting of melanoma cells, basal cell carcinoma cells,squamous cell carcinoma cells, neuroblastoma cells, glioblastomamultiforme cells, myeloid leukemic cells, breast carcinoma cells, coloncarcinoma cells, endometrial carcinoma cells, lung carcinoma cells,ovarian carcinoma cells, prostate carcinoma cells, cervical carcinomacells, osteosarcoma cells and lymphoma cells.
 38. A method foridentifying a compound capable of inducing specific patterns of DNAdamage caused by UV irradiation and gamma irradiation in human melanomacells comprising: a) incubating an appropriate concentration of thehuman melanoma cells with an appropriate concentration of the compound;and b) measuring the expression of mda-9, the altered expression ofmda-9 gene indicating that the compound is capable of inducing specificpatterns of DNA damage caused by UV irradiation and gamma irradiation inhuman melanoma cells.
 39. A method for identifying a temporallyexpressed gene from cancer cells induced to undergo apoptosis by achemotherapeutic agent, comprising: a) incubating an appropriateconcentration of the cancer cells with an appropriate concentration ofthe chemotherapeutic agent; and b) measuring the expression of mda-9,the modified expression of mda-9 gene indicating that the compound iscapable of inducing the cancer cells to undergo apoptosis.
 40. A methodof claim 39, wherein the cancer cells are selected from a groupconsisting of melanoma cells, basal cell carcinoma cells, squamous cellcarcinoma cells, neuroblastoma cells, glioblastoma multiforme cells,myeloid leukemic cells, breast carcinoma cells, colon carcinoma cells,endometrial carcinoma cells, lung carcinoma cells, ovarian carcinomacells, prostate carcinoma cells, cervical carcinoma cells, osteosarcomacells and lymphoma cells.
 41. A method for identifying a compoundcapable of elevating mda-9 expression in cancer cells comprising: a)incubating an appropriate concentration of the cancer cells with anappropriate concentration of the compound; b) measuring the expressionof mda-9 to determine whether the expression of the mda-9 gene iselevated.
 42. A method of claim 41, wherein the compound capable ofelevating mda-9 expression in cancer cells is IFN-γ.
 43. A method ofclaim 41, wherein the compound capable of elevating mda-9 expression incancer cells is a cytokine.
 44. A method of claim 43, wherein thecytokine is selected from a group consisting of IFN-α, IFN-β, IFN-γ,TNF-α, stem cell growth factors, colony stimulating factor, GMCSF, andinterleukins, including interleukin-6.
 45. A method of claim 41, whereinthe cancer cells are selected from a group consisting of human melanomacells, basal cell carcinoma cells, squamous cell carcinoma cells,neuroblastoma cells, glioblastoma multiforme cells, myeloid leukemiccells, breast carcinoma cells, colon carcinoma cells, endometrialcarcinoma cells, lung carcinoma cells, ovarian carcinoma cells, prostatecarcinoma cells, cervical carcinoma cells, osteosarcoma cells andlymphoma cells.
 46. An antisense oligonucleotide having a sequencecapable of specifically hybridizing to an mRNA molecule encoding a humanmda-9 protein so as to prevent expression of the mRNA molecule.
 47. Anantisense oligonucleotide having a sequence capable of specificallyhybridizing to an mRNA molecule encoding a human mda-9 protein so as toprevent translation of the mRNA molecule.
 48. An antisenseoligonucleotide having a sequence capable of specifically hybridizing tothe promoter of the isolated nucleic acid molecule of either of claims32 or 33, thereby preventing transcription.
 49. An antisenseoligonucleotide having a sequence capable of specifically hybridizing tothe mRNA of the isolated nucleic acid molecule of either of claims 32 or33 and capable of, degrading the hybridized mRNA.
 50. A purified mda-9protein.
 51. A purified human mda-9 protein of claim 50 having an aminoacid sequence as set forth in FIG.
 7. 52. An antibody directed to apurified mda-9 protein of either of claims 50 or
 51. 53. An antibodycapable of specifically recognizing the mda-9 protein of either ofclaims 50 or
 51. 54. An antibody of either of claims 52 or 53, whereinthe mda-9 protein is a human mda-9 protein.
 55. An monoclonal orpolyclonal antibody of any one of claims 52, 53, and
 54. 56. Apharmaceutical composition comprising an amount of the oligonucleotideof any one of claims 46, 47, 48, and 49 effective to prevent expressionof a human mda-9 protein and a pharmaceutically acceptable carrier. 57.A method of treating cancer in a subject by administering thepharmaceutical composition of claim 56, thereby treating cancer in asubject.
 58. A method of claim 57, wherein the cancer is selected from agroup consisting of human melanoma, basal cell carcinoma, squamous cellcarcinoma, neuroblastoma, glioblastoma multiforme carcinoma, myeloidleukemia, breast carcinoma, colon carcinoma, endometrial carcinoma, lungcarcinoma, ovarian carcinoma, prostate carcinoma, cervical carcinoma,osteosarcoma and lymphoma.
 59. A method of claim 57, wherein theexpression of a human mda-9 protein is prevented by hybridization of theantisense oligonucleotide to the mda-9 gene promoter or mda-9 mRNAmolecules regulated by a tissue specific promoter that permitsexpression of the human mda-9 antisense sequence only in the cancercells.
 60. A method of claim 59, wherein the cancer is melanoma and thetissue specific promoter is a tyrosinase promoter.
 61. A pharmaceuticalcomposition of claim 56 further comprising a substance which facilitatesthe delivery of said oligonucleotide into the cell.
 62. A pharmaceuticalcomposition of claim 61, wherein the substance which facilitates thedelivery of the oligonucleotide into the cell is a liposome or anantibody.
 63. A pharmaceutical composition of claim 61, wherein theoligonucleotide is delivered into the cell by a viral vector.
 64. Amethod of inhibiting expression of a mda-9 gene in a subject comprisingintroducing a vector containing a nucleic acid molecule which rendersthe mda-9 gene functionless into the subject under conditions permittingthe inhibition of the expression of the mda-9 gene.
 65. A method ofclaim 64, wherein the nucleic acid is an antisense oligonucleotidehaving a sequence capable of specifically hybridizing to an mRNAmolecule encoding a human mda-9 protein.
 66. A method of claim 64,wherein the nucleic acid contains a mutation or deletion of the mda-9gene having the appropriate flanking sequences.
 67. A method of treatinga cancer in a subject by administering a pharmaceutical compositioncomprising an effective amount of the antibody of either of claims 52 or53, thereby treating the cancer in a subject.
 68. A method of claim 65,wherein the cancer is a melanoma.
 69. A method of increasing theexpression of mda-9 to inhibit cell growth comprising transfecting cellswith an expression vector comprising an mda-9 gene insert to induceexpression of mda-9 in cells thereby inhibiting growth of the cells. 70.The method of claim 69, wherein the mda-9 gene insert is in either thesense or antisense orientation.
 71. The method of claim 69, whereinmda-9 gene insert is in the sense orientation and mda-9 isoverexpressed.
 72. The method of claim 69, wherein mda-9 gene insert isin the antisense orientation and mda-9 expression is inhibited.
 73. Themethod of claim 69, wherein the cells are selected from the groupconsisting of human melanoma, basal cell carcinoma, squamous cellcarcinoma, neuroblastoma, glioblastoma multiforme carcinoma, myeloidleukemia, breast carcinoma, colon carcinoma, endometrial carcinoma, lungcarcinoma, ovarian carcinoma, prostate carcinoma, cervical carcinoma,osteosarcoma and lymphoma.
 74. A method of treating a cancer in asubject by increasing mda-9 expression in cancer cells of the subject-to induce partial differentiation in the cancer cells by administering apharmaceutical composition comprising a targeting vector and an agentwhich partially induces differentiation.
 75. The method of claim 74,wherein the targeting vector is an mda-9 expression vector and saidvector is selected from the group consisting of an adenovirus, anadeno-associated virus, a retrovirus, a vaccinia virus, and an EpsteinBarr virus.
 76. The method of claim 74, wherein the agent whichpartially induces differentiation is a cytokine, a DNA damagingchemotherapeutic agent, or a physical therapeutic agent.
 77. The methodof claim 76, wherein the cytokine is selected from the group consistingof IFN-α, IFN-β, IFN-γ and TNF-α.
 78. The method of claim 76, whereinthe DNA damaging chemotherapeutic agent is selected from the groupconsisting of dactinomycin, cis-platinum, and taxol and its analogs. 79.The method of claim 76, wherein the physical therapeutic agent is gammairradiation.
 80. The method of claim 74, wherein the cancer cells areselected from the group consisting of human melanoma, basal cellcarcinoma, squamous cell carcinoma, neuroblastoma, glioblastomamultiforme carcinoma, myeloid leukemia, breast carcinoma, coloncarcinoma, endometrial carcinoma, lung carcinoma, ovarian carcinoma,prostate carcinoma, cervical carcinoma, osteosarcoma and lymphoma.
 81. Amethod of treating a cancer in a subject by increasing mda-9 expressionin cancer cells of the subject to suppress growth of the cancer cells byadministering a pharmaceutical composition comprising a targeting vectorand an agent which partially induces differentiation.
 82. The method ofclaim 81, wherein the targeting vector is an mda-9 expression vector andsaid vector is selected from the group consisting of an adenovirus, anadeno-associated virus, a retrovirus, a vaccinia virus, and an EpsteinBarr virus.
 83. The method of claim 81, wherein the agent whichpartially induces differentiation is a cytokine, a DNA damagingchemotherapeutic agent, or a physical therapeutic agent.
 84. The methodof claim 83, wherein the cytokine is selected from the group consistingof IFN-α, IFN-β, IFN-γ and TNF-α.
 85. The method of claim 83, whereinthe DNA damaging chemotherapeutic agent is selected from the groupconsisting of dactinomycin, cis-platinum, and taxol and its analogs. 86.The method of claim 83, wherein the physical therapeutic agent is gammairradiation.
 87. The method of claim 81, wherein the cancer cells areselected from the group consisting of human melanoma, basal cellcarcinoma, squamous cell carcinoma, neuroblastoma, glioblastomamultiforme carcinoma, myeloid leukemia, breast carcinoma, coloncarcinoma, endometrial carcinoma, lung carcinoma, ovarian carcinoma,prostate carcinoma, cervical carcinoma, osteosarcoma and lymphoma.
 88. Acell having an exogenous indicator gene under the control of theregulatory element of a mda-9 gene.
 89. The cell of claim 88, whereinthe cell is a normal cell.
 90. The cell of claim 88, wherein the cell isa cancer cell.
 91. The cell of any of claim 88, 89, or 90, wherein theindicator gene codes for beta-galactosidase, luciferase, chloramphenicoltransferase or secreted alkaline phosphatase.
 92. A method fordetermining whether an agent is capable of modifying DNA damage andrepair pathways, differentiation, apoptosis or operates through acytokine modulatory pathway comprising contacting an amount of the agentwith the cell of claim 88, wherein a change in expression of theindicator gene compared to the expression in control cells indicatesthat the agent modifies DNA damage and repair pathways, differentiation,apoptosis or operates through a cytokine modulatory pathway.
 93. Themethod of claim 92, wherein the change in expression is either adecrease in expression or an increase in expression of the indicatorgene.
 94. A nucleic acid molecule comprising a sequence of the promoterof an mda-9 gene protein.
 95. The method of claim 92, wherein the agentis a small molecule selected from a recombinatorial library, a peptidelibrary, a peptide-derived library or a chemical library.